p21 peptides

ABSTRACT

The present invention relates to p21 derived peptides capable of inhibiting CDK/cyclin complexes, particularly cyclins A or E/CDK2, by modifying the interaction with their substrates. The peptides are derived from a C-terminal region of p21 and display selectivity for cyclin/CDK2 inhibition over cyclin/CDK4 inhibition. Variants of such peptides particularly involving certain alanine replacements are shown to be particularly potent.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. Ser. No. 09/726,470, filed Nov. 29, 2000, and claims priority to Great Britain application Serial No. 9928323.6 filed Nov. 30, 1999, the contents each of which are entirely incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to substances and their therapeutic use, and in particular to specific regions of p21^(WAF1) that bind to G1 and S phase specific cyclins, preferably ones activating CDK2 and to substances and mimetics based on this region. The invention also relates to assay methods and means for identifying substances useful for interfering with protein-protein interactions involving cyclins, particularly CDK/cyclin interactions and preferably capable of inhibiting CDK2 activity.

[0003] p21^(WAF1) is an inhibitor of both the G1 cyclin dependent protein kinases (CDKs; which control the progression from G1 into S phase) (Harper et al., 1995) and proliferating cell nuclear antigen (PCNA; an essential DNA-replication factor) (Florez-Rozas et al., 1994; Waga et al., 1994). Thus, inhibition of the function of either CDKs or PCNA provides, in theory, two distinct avenues for drug discovery based on the activity of p21^(WAF1). The PCNA binding function of p21^(WAF1) can be mimicked by a 20-amino acid peptide derived from the C-terminal domain of p21^(WAF1) and this peptide is sufficient partially to inhibit SV40 replication in vitro (Warbrick et al., 1995).

[0004] Despite its PCNA binding role, the primary function of the p21^(WAF1) protein as a growth suppressor appears to be inhibition of the G1 cyclin-CDK complexes (Chen et al., 1995; Harper et al., 1995; Luo et al., 1995; Nakanishi et al., 1995b). Luo et al. (1995) reported the N-terminal domain of p21 composed of residues 1-75, to act as a CDK-inhibitor in vitro, inhibiting cyclin E-CDK2.

[0005] WO 97/42222 (Cyclacel Ltd) discloses peptide fragments of p21^(WAF1) that interact with CDK4/cyclin D1. Thus it was observed that p21₍₁₆₋₃₅₎ and p21₍₄₆₋₆₅₎ bind to CDK4 and cyclin D1 respectively. Of these, only p21₍₁₆₋₃₅₎ was observed to inhibit CDK activity. p21₍₁₄₁₋₁₆₀₎ was observed to bind to CDK4 and cyclin D1 and to be a potent inhibitor of CDK4.

[0006] This data supported the known phenomenon of peptides including the sequence LFG as being the binding motif essential for the interaction of the p21 family with cyclins [Chen J et al.(1996), Lin J et al. and Russo A A et al.] and the further known properties of the amino-terminal half of p21 as being required for binding to CDK complex.

[0007] It should be borne in mind when considering the prior art discussed herein that unless otherwise explicitly stated the references to “motifs” is made with reference to papers that have made deductions and predictions based upon the activity of longer peptides usually consisting of at least 12 amino acids. Thus, the motifs are no more than conjecture based upon the a specific set of reactions. Such motifs provide no indication as to the actual length of peptide or modifications that could be made to retain and/or even enhance activity or specificity.

[0008] The sequence p21₍₁₄₁₋₁₆₀₎ (disclosed in WO97/42222 and Ball K. et al) in respect of cyclin D1/CDK4 inhibition was subjected to analysis in order to determine the minimum length of an inhibitory peptide upon which novel antiproliferative drugs could be designed.

[0009] Observations of CDK4/cyclin D1 inhibitory activity led to the identification of an inhibitory motif comprising RRLIF (p21₍₁₅₅₋₁₅₉₎₎ (SEQ ID No. 5), the bold residues being described as essential for activity and the underlined residue contributing towards inhibitory activity. Further observations in these disclosures include the retention of inhibitory activity against cyclin D1-CDK4 by the peptide KRRLIFSK (p21₍₁₅₄₋₁₆₁₎₎ (SEQ ID No. 6) albeit at a concentration 1000 times greater than the parent sequence p21₁₄₁₋₁₆₀ and that the substitution of aspartic acid at position 149 of p21₁₄₁₋₁₆₀ by alanine surprisingly reduced the IC₅₀ of the full length peptide from 100 nM to 46 nM. Thus, although identifying the RRLIF (SEQ ID No. 5) motif as being important to cyclinD1/CDK4 inhibition, Ball et al. is inconclusive as to the actual minimum length peptide required for enhanced activity. The effect of the Asp149 to Ala substitution has not proven reproducible.

[0010] In summary, WO97/42222 and Ball et al teach that there are sequences within the carboxy terminal region of p21 that are capable of interacting with CDK4/cyclin D in a manner that is inhibitory to CDK4 and further involves specific binding to cyclin D. Though the peptide p21₍₁₄₁₋₁₆₀₎ is described as being preferred, an 8-mer comprising p21₍₁₅₄₋₁₆₁₎ (KRRLIFSK) (SEQ ID No. 6) was inhibitory, but at higher concentrations. Finally, alanine replacement at position 149 within p21₁₄₁₋₁₆₀ increased the inhibitory activity.

[0011] Thus, although the art indicates that this is an interesting region of p21 to investigate, no guidance is provided as to the identity of further fragments that would be preferably active against CDK4/cyclin D or any other CDK/cyclin enzymes.

[0012] Chen J et al. (Mol Cell Biol (1996) 16(9) 4673-4682) disclose a 12-mer corresponding to p21₁₇₋₂₄ as being a cyclin binding domain of p21. They further identify a less avid cyclin binding region as p21₁₅₀₋₁₆₁. Mutation and inhibition analysis demonstrated that the principal site of interaction with cyclin A was p21₁₇₋₂₄, being a better inhibitor than p21₁₅₀₋₁₆₁ consistent with its greater avidity for cyclins such that it can be detected by pull-down assay. Interaction of p21₁₅₀₋₁₆₁ could only “be inferred from competition for binding and kinase inhibition assays. The importance of the p21₁₅₀₋₁₆₁ in vivo was questioned due to the possibility of the relevant site being occupied by PCNA.

[0013] Adams D A et al. (Mol Cell Biol (1996) 16(12) 6623-6633) discloses N- and C-terminal regions of p21 that putatively bind to CDK2/cyclin. A 14-mer (p21₁₄₉₋₁₆₂) is disclosed as inhibiting the binding of cyclin A to E2F1 and the binding of cyclins A and E to GST-p21. An amino acid sequence containing 8 amino acid residues (PVKRRLDL) (SEQ ID No. 7), derived from the transcription factor E2F1 was shown to bind to cyclin A/E-CDK2 complexes. An alanine scan of the 8-mer identified, on a qualitative level that certain modified forms of the peptide retained this activity. Noteworthy is that deletion or alanine replacement of either terminal amino acid reduced or abolished the ability to compete with GST-E2F1 for cyclin A binding.

[0014] In a further paper, Adams D A et al. (Mol Cell Biol (1999) 19(2) 1068-1080) investigated the existence of an E2F1-like motif within pRB as a means to explain its interaction with cyclin A/CDK2. A single 10-mer, pRB869-878 was the shortest pRB derived peptide investigated.

[0015] In a subsequent paper, Chen et al. (Proc. Natn. Acad. Sci. (1999) 96, 4325-4329) disclosed two E2F1 derived 8-mers as possessing the ability to interact with the cyclin A/CDK2 complex, being PVKRRLFG (SEQ ID No. 8) and PVKRRLDL (SEQ ID No. 7). These peptides were tested in whole cell assays using membrane translocation carrier peptides HIV-TAT or Penetratin®.

[0016] Brown N R et al. (Nature Cell Biol. (1999) 1, 438-443) describe a crystal structure of the cyclin A3/phospho-CDK2 complex with an 11-mer derived from p107 including the RXLF SEQ ID No. 9) motif. Of the 11-mer, the region RRLFGE (SEQ ID No. 10), was found to be within the binding region of cyclin A forming interactions with M210, I213, W217, E220, L253 and Q254.

[0017] An aim of the present invention has been to identify further peptides derived from p21 that retain or improve upon the inhibitory activities described in the art, particularly with regard to substrate specificity and peptide chain length as described in detail below.

SUMMARY OF THE INVENTION

[0018] A first aspect of the present invention therefore relates to a p21 derived peptide of formula; DFYHSKRRLIF (SEQ ID No. 1) or such a peptide (i) bearing a further amino acid residue at either end; or,(ii) having up to 7 amino acid residues deleted from the N-terminal end; and variants thereof wherein at least one amino acid residue is replaced by an alternative natural or unnatural replacement amino acid residue, with the proviso that the motif XLXF (SEQ ID No. 11) is retained. The peptide of SEQ ID No. 1 corresponds to p21(149-159). In an embodiment of this aspect up to 5 amino acid residues are deleted from the N-terminal and the motif RXLXF (SEQ ID No. 12) is retained.

[0019] A second aspect of the present invention relates to a p21 derived peptide of formula;X₁X₂X₃RX₄LX₅F (SEQ ID No. 2) wherein X₁, X₃, X₄ and X₅ are any amino acid and X₂ is serine or alanine; and variants thereof.

[0020] In one aspect, the invention pertains to a peptide of formula I: N₁DFYHSKRRLIFN₂ (formula I) (SEQ ID No. 4), comprising the motif XLXF (SEQ ID No. 11) wherein N₁ and N₂ are independently a natural or non-natural amino acid or nothing; or the peptide of formula I having up to 8 amino acid residues deleted from the N-terminal end; and variants thereof wherein at least one amino acid residue is replaced by an alternative natural or non-natural replacement amino acid residue, with the proviso that the motif XLXF(SEQ ID No. 11) is retained, wherein X refers to any natural or unnatural amino acid.

[0021] In one embodiment, N₁ and N₂ are independently selected from nothing and the polar residues C, N, Q, S, T and Y.

[0022] In one embodiment, N₁ is a natural or unnatural amino acid. In another embodiment, N₁ is threonine.

[0023] In one embodiment, N₂ is a natural or unnatural amino acid. In one embodiment, N₁ is serine.

[0024] In another embodiment, up to 6 amino acid residues are deleted from the N-terminal end of the peptide of formula I.

[0025] In another embodiment, from 3-5 amino acid residues are deleted from the N-terminal end of the peptide of formula I.

[0026] In yet another embodiment 4 amino acid residues are deleted from the N-terminal end of the peptide of formula I.

[0027] In one embodiment, N₂ is a natural or unnatural amino acid.

[0028] In another embodiment, N₂ is serine.

[0029] In yet another embodiment, 7 or 8 amino acid residues are deleted from the N-terminal end of the peptide of formula I.

[0030] In another aspect, the invention pertains to a peptide of formula DFYHSKRRLIF (SEQ ID No. 1), comprising the motif XLXF (SEQ ID No. 11), or such a peptide (i) bearing a further amino acid residue at either end; and, (ii) having up to 7 amino acid residues deleted from the N-terminal end; and variants thereof wherein at least one amino acid residue is replaced by an alternative natural or unnatural replacement amino acid residue, with the proviso that the motif XLXF (SEQ ID No. 11) is retained, wherein the peptide of SEQ ID No. 1 is modified by at least one of; deletion, addition or substitution of one or more amino acid residues, or by substitution of one or more natural amino acid residues by the corresponding D-stereomer or by a non-natural amino acid residue, chemical derivatives of the peptides, cyclic peptides derived from the peptides or from the peptide derivatives dual peptides, multimers of the peptides and any of said peptides in the D-stereomer form, or the order of the final two residues at the C-terminal end are reversed.

[0031] In one embodiment, the serine residue corresponding to p21(153Ser), is replaced by an alanine residue.

[0032] In another embodiment, a peptide is selected from; D F Y H S K R R L I F S, (SEQ. ID NO: 13) T D F Y H S K R R L I F,, (SEQ ID NO: 14) A F Y H S K R R L I F S, (SEQ ID NO: 15) D A Y H S K R R L I F S, (SEQ ID NO: 16) D F A H S K R R L I F S,, (SEQ ID NO: 17) D F Y A S K R R L I F S, (SEQ ID NO: 18) D F Y H A K R R L I F S, (SEQ ID NO: 19) D F Y H S A R R L I F S, (SEQ ID NO: 20) D F Y H S K R A L I F S, (SEQ ID NO: 21) D F Y H S K R R L A F S, (SEQ ID NO: 22) D F Y H S K R R L I F A, (SEQ ID NO: 23) F Y H S K R R L I F S, (SEQ ID NO: 24) Y H S K R R L I F S, (SEQ ID NO: 25) H S K R R L I F S, (SEQ ID NO: 26) D F Y H S K R R L I F, (SEQ ID NO: 1) F Y H S K R R L I F, (SEQ ID NO: 27) Y H S K R R L I F, (SEQ ID NO: 28) H S K R R L I F, (SEQ ID NO: 29) S K R R L I F, (SEQ ID NO: 30) K R R L I F, (SEQ ID NO: 31) H- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 32) H- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 33) H- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 34) H- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 35) H- His- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 36) H- Asn- Leu- Phe- Gly —NH2 (SEQ ID NO. 37) H- Arg- Asn- Leu- Phe- Gly —NH2 (SEQ ID NO. 38) H- Abu- Arg- Asn- Leu- Phe- Gly —NH2 (SEQ ID NO. 39) H- Ala- Abu- Arg- Asn- Leu- Phe- Gly —NH2 and (SEQ ID NO. 40) H- Ser- Ala- Abu- Arg- Asn- Leu- Phe- Gly —NH2 (SEQ ID NO. 41)

[0033] In still another aspect, the invention pertains to a peptide of formula II: X₁X₂X₃RX₄LX₅F (formula II) (SEQ ID No. 2) wherein X₁, X₃, X₄ and X₅ may be any amino acid and X₂ is serine or alanine; and variants thereof.

[0034] In one embodiment, X₅ is selected from isoleucine and glycine.

[0035] In one embodiment, X₁ and X₄ are both basic amino acid residues and X₃ is a basic or polar residue.

[0036] In one embodiment, X₁ is histidine and X₄ is arginine, and X₃ is lysine or cysteine.

[0037] In another aspect the invention pertains to a peptide of formula: X₁X₂X₃RX₄LX₅F (SEQ ID No. 2) wherein X₁, X₃, X₄ and X₅ may be any amino acid and X₂ is serine or alanine; and variants thereof, wherein the peptide is modified by at least one of a deletion, addition or substitution of one or more amino acid residues, or by substitution of one or more natural amino acid residues by the corresponding D-stereomer or by a non-natural amino acid residue, chemical derivatives of the peptides, cyclic peptides derived from the peptides or from the peptide derivatives, dual peptides, multimers of the peptides and any of said peptides in the D-stereomer form, or the order of the final two residues at the C-terminal end are reversed.

[0038] In yet another aspect, the invention pertains to a peptide of formula: X₁X₂X₃RX₄LX₅F (SEQ ID No. 2) wherein X₁, X₃, X₄ and X₅ may be any amino acid and X₂ is serine or alanine; and variants thereof, wherein: (a) X₁ is deleted or is any amino acid, (b) X₂ is serine or alanine or a straight or branched chain amino acid, (c) X₃ is a basic amino acid or straight chain aliphatic amino acid, (d) R is unchanged or conservatively substituted (by basic amino acids), (e) X₄ is any amino acid that is capable of providing at least one site for participating in hydrogen bonding, (f) L is unchanged or conservatively substituted, (g) X₅ is any amino acid, or (h) F is unchanged or substituted by any aromatic amino acid.

[0039] In another aspect, the invention pertains to a peptide of formula: X₁X₂X₃RX₄LX₅F (SEQ ID No. 2), wherein (a) X₁ is histidine, deleted or replaced by a natural or unnatural amino acid residue-such as alanine, 3-pyridylalanine (Pya), 2-thienylalanine (Thi), homoserine (Hse), phenylalanine, or diaminobutyric acid (Dab), (b) X₂ is alanine or an alternative natural or unnatural amino acid residue having a smaller or slightly larger aromatic or aliphatic side chain, such as glycine, aminobutyric acid (Abu), norvaline (Nva), t-butylglycine(Bug), valine, isoleucine, phenylglycine (Phg) or phenylalanine, (c) X₃ is lysine or either a basic residue such as arginine or an uncharged natural or unnatural amino acid residue, such as norleucine (Nle), aminobutyric acid (Abu) or leucine, (d) arginine is replaced by either a basic residue such as lysine or an uncharged natural or unnatural amino acid residue, such as citrulline (Cit), homoserine, histidine, norleucine (Nle) or glutamine, (e) X₄ is arginine or a natural or unnatural amino acid residue, such as asparagine, proline, serine, aminoisobutyric acid (Aib) or sarcosine (Sar), or an amino acid residue capable of forming a cyclic linkage such as lysine or ornithine, (f) leucine is replaced with a natural or unnatural amino acid residue having a slightly larger aromatic or aliphatic side chain, such as norleucine, norvaline, cyclohexylalanine (Cha), phenylalanine or 1-naphthylalanine (1 Nal), (g) X₅ is isoleucine or an alternative natural or unnatural amino acid residue having a slightly larger aromatic or aliphatic side chain, such as norleucine, norvaline, cyclohexylalanine (Cha), phenylalanine or 1-naphthylalanine (1 Nal), (h) phenylalanine is replaced with a natural or unnatural amino acid such as leucine, cyclohexylalanine (Cha), homophenylalanine (Hof), tyrosine, para-fluorophenylalanine (pFPhe), meta-fluorophenylalanine (mFPhe), trptophan, 1-naphthylalanine (1Nal), 2-naphthylalanine (2Nal), biphenylalanine (Bip) or (Tic), (i) X₅ and the terminal phenylalanine residue are reversed, or (j)the peptide is in cyclic form by the formation of a linkage between the side chain of X₄ and the C-terminus residue.

[0040] In one embodiment, X₂ is alanine.

[0041] In one embodiment, X₅ is isoleucine.

[0042] In another embodiment, a peptpide is selected from the group consisting of: H S K R R L I F, (SEQ ID NO. 29) H A K R R L I F, (SEQ ID NO. 42) H S K R R L F G, (SEQ ID NO. 43) H A K R R L F G, (SEQ ID NO. 44) K A C R R L F G, (SEQ ID NO. 45) K A C R R L I F, (SEQ ID NO. 46) X1 X2 X3 R X4 L X5 F (SEQ ID NO. 2) H- His- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 36) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 47) H- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 48) H- Pya- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 49) H- Thi- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 50) H- Hse- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 51) H- Phe- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 52) H- Dab- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 53) H- His- Gly- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 54) H- His- Abu- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 55) H- His- Nva- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 56) H- His- Bug- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 57) H- His- Val- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 58) H- His- Ile- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 59) H- His- Phg- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 60) H- His- Phe- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 61) H- His- Ala- Ala- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 62) H- His- Ala- Nle- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 63) H- His- Ala- Abu- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 64) H- His- Ala- Leu- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 65) H- His- Ala- Arg- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 66) H- His- Ala- Lys- Ala- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 67) H- His- Ala- Lys- Cit- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 68) H- His- Ala- Lys- Hse- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 69) H- His- Ala- Lys- His- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO  70) H- His- Ala- Lys- Nle- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 71) H- His- Ala- Lys- Gln- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 72) H- His- Ala- Lys- Lys- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 73) H- His- Ala- Lys- Arg- Ala- Leu- Ile- Phe —NH2 (SEQ ID NO. 74) H- His- Ala- Lys- Arg- Asn- Leu- Ile- Phe —NH2 (SEQ ID NO. 75) H- His- Ala- Lys- Arg- Pro- Leu- Ile- Phe —NH2 (SEQ ID NO. 76) H- His- Ala- Lys- Arg- Ser- Leu- Ile- Phe —NH2 (SEQ ID NO. 77) H- His- Ala- Lys- Arg- Aib- Leu- Ile- Phe —NH2 (SEQ ID NO. 78) H- His- Ala- Lys- Arg- Sar- Leu- Ile- Phe —NH2 (SEQ ID NO. 79) H- His- Ala- Lys- Arg- Cit- Leu- Ile- Phe —NH2 (SEQ ID NO. 80) H- His- Ala- Lys- Arg- Arg- Ala- Ile- Phe —NH2 (SEQ ID NO. 81) H- His- Ala- Lys- Arg- Arg- leu- Ile- Phe —NH2 (SEQ ID NO. 82) H- His- Ala- Lys- Arg- Arg- Ile- Ile- Phe —NH2 (SEQ ID NO. 83) H- His- Ala- Lys- Arg- Arg- Val- Ile- Phe —NH2 (SEQ ID NO. 84) H- His- Ala- Lys- Arg- Arg- Nle- Ile- Phe —NH2 (SEQ ID NO. 85) H- His- Ala- Lys- Arg- Arg- Nva- Ile- Phe —NH2 (SEQ ID NO. 86) H- His- Ala- Lys- Arg- Arg- Cha- Ile- Phe —NH2 (SEQ ID NO. 87) H- His- Ala- Lys- Arg- Arg- Phe- Ile- Phe —NH2 (SEQ ID NO. 88) H- His- Ala- Lys- Arg- Arg- 1Nap- Ile- Phe —NH2 (SEQ ID NO. 89) H- His- Ala- Lys- Arg- Arg- Leu- Ala- Phe —NH2 (SEQ ID NO. 90) H- His- Ala- Lys- Arg- Arg- Leu- Leu- Phe —NH2 (SEQ ID NO. 91) H- His- Ala- Lys- Arg- Arg- Leu- Val- Phe —NH2 (SEQ ID NO. 92) H- His- Ala- Lys- Arg- Arg- Leu- Nle- Phe —NH2 (SEQ ID NO. 93) H- His- Ala- Lys- Arg- Arg- Leu- Nva- Phe —NH2 (SEQ ID NO. 94) H- His- Ala- Lys- Arg- Arg- Leu- Cha- Phe —NH2 (SEQ ID NO. 95) H- His- Ala- Lys- Arg- Arg- Leu- Phe- Phe —NH2 (SEQ ID NO. 96) H- His- Ala- Lys- Arg- Arg- Leu- 1Nap- Phe —NH2 (SEQ ID NO. 97) H- His- Ala- Lys- Arg- Arg- Leu- Phe —NH2 (SEQ ID NO. 98) H- His- Ala- Lys- Arg- Arg- Leu- Ile- Leu —NH2 (SEQ ID NO. 99) H- His- Ala- Lys- Arg- Arg- Leu- Ile- Cha —NH2 (SEQ ID NO. 100) H- His- Ala- Lys- Arg- Arg- Leu- Ile- Hof —NH2 (SEQ ID NO. 101) H- His- Ala- Lys- Arg- Arg- Leu- Ile- Tyr —NH2 (SEQ ID NO. 102) H- His- Ala- Lys- Arg- Arg- Leu- Ile- pFPhe —NH2 (SEQ ID NO. 103) H- His- Ala- Lys- Arg- Arg- Leu- Ile- mFPhe —NH2 (SEQ ID NO. 104) H- His- Ala- Lys- Arg- Arg- Leu- Ile- Trp —NH2 (SEQ ID NO. 105) H- His- Ala- Lys- Arg- Arg- Leu- Ile- 1Nap —NH2 (SEQ ID NO. 106) H- His- Ala- Lys- Arg- Arg- Leu- Ile- 2Nap —NH2 (SEQ ID NO. 107) H- His- Ala- Lys- Arg- Arg- Leu- Ile- Lys —NH2 (SEQ ID NO. 108) H- His- Ala- Lys- Arg- Arg- Leu- Ile- Tic —NH2 (SEQ ID NO. 109) H- His Ala Lys Arg Arg Leu Ile L-Pse OH (SEQ ID NO. 110) H- His Ala Lys Arg Arg Leu Ile D-Pse OH (SEQ ID NO. 111) H- His Ser Lys Arg Arg Leu Ile L-Pse OH (SEQ ID NO. 112) H- His Ser Lys Arg Arg Leu Ile D-Pse OH (SEQ ID NO. 113) H- His Ala Lys Arg Arg Leu Ile L-Psa OH (SEQ ID NO. 114) H- His Ala Lys Arg Arg Leu Ile D-Psa OH (SEQ ID NO. 115) H- His Ser Lys Arg Arg Leu Ile L-Psa OH (SEQ ID NO. 116) H- His Ser Lys Arg Arg Leu Ile D-Psa OH (SEQ ID NO. 117) H- His Ala Lys Arg Arg Leu Ile Dhp OH (SEQ ID NO. 118) H- His Ser Lys Arg Arg Leu Ile Dhp OH (SEQ ID NO. 119) H- His Ala Lys Arg Arg Leu Ile Pheol (SEQ ID NO. 120) H- His Ser Lys Arg Arg Leu Ile Pheol (SEQ ID NO. 121) H- Ala- Ala- Abu- Arg- Arg- Leu- Ile- pFPhe —NH2 (SEQ ID NO. 122) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- pFPhe —NH2 (SEQ ID NO. 123) H- Ala- Ala- Lys- Arg- Cit- Leu- Ile- pFPhe —NH2 (SEQ ID NO. 124) H- Ala- Ala- Lys- Arg- Arg- Leu- Ala- pFPhe —NH2 (SEQ ID NO. 125) H- Ala- Ala- Abu- Arg- Ser- Leu- Ile- pFPhe —NH2 (SEQ ID NO. 126) H- Ala- Ala- Lys- Gln- Arg- Leu- Ile- pFPhe —NH2 (SEQ ID NO. 127) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- pFPhe —NH2 (SEQ ID NO. 128) H- Gly- Ala- Lys- Arg- Arg- Leu- Ile- pFPhe —NH2 (SEQ ID NO. 129) H- Ala- Ala- Lys- hArg- Arg- Leu- Ile- pFPhe —NH2 (SEQ ID NO. 130) H- Ala- Ala- Lys- Ser- Arg- Leu- Ile- pFPhe —NH2 (SEQ ID NO. 131) H- Ala- Ala- Lys- Hse- Arg- Leu- Ile- pFPhe —NH2 (SEQ ID NO. 132) H- Ala- Ala- Lys- Arg- Lys- Leu- Ile- pFPhe —NH2 (SEQ ID NO. 133) H- Ala- Ala- Lys- Arg- Orn- Leu- Ile- pFPhe —NH2 (SEQ ID NO. 134) H- Ala- Ala- Lys- Arg- Gln- Leu- Ile- pFPhe —NH2 (SEQ ID NO. 135) H- Ala- Ala- Lys- Arg- Hse- Leu- Ile- pFPhe —NH2 (SEQ ID NO. 136) H- Ala- Ala- Lys- Arg- Thr- Leu- Ile- pFPhe —NH2 (SEQ ID NO. 137) H- Ala- Ala- Lys- Arg- Nva- Leu- Ile- pFPhe —NH2 (SEQ ID NO. 138) H- Ala- Ala- Lys- Arg- Arg- Phg- Ile- pFPhe —NH2 (SEQ ID NO. 139) H- Ala- Ala- Lys- Arg- Arg- Met- Ile- pFPhe —NH2 (SEQ ID NO. 140) H- Ala- Ala- Lys- Arg- Arg- Ala- Ile- pFPhe —NH2 (SEQ ID NO. 141) H- Ala- Ala- Lys- Arg- Arg- Hof- Ile- pFPhe —NH2 (SEQ ID NO. 142) H- Ala- Ala- Lys- Arg- Arg- hLeu- Ile- pFPhe —NH2 (SEQ ID NO. 143) H- Ala- Ala- Lys- Arg- Arg- aIle- Ile- pFPhe —NH2 (SEQ ID NO. 144) H- Ala- Ala- Lys- Arg- Arg- Leu- Gly- pFPhe —NH2 (SEQ ID NO. 145) H- Ala- Ala- Lys- Arg- Arg- Leu- βAla pFPhe —NH2 (SEQ ID NO. 146) H- Ala- Ala- Lys- Arg- Arg- Leu- Phg- pFPhe —NH2 (SEQ ID NO. 147) H- Ala- Ala- Lys- Arg- Arg- Leu- Aib- pFPhe —NH2 (SEQ ID NO. 148) H- Ala- Ala- Lys- Arg- Arg- Leu- Sar- pFPhe —NH2 (SEQ ID NO. 149) H- Ala- Ala- Lys- Arg- Arg- Leu- Pro- pFPhe —NH2 (SEQ ID NO. 150) H- Ala- Ala- Lys- Arg- Arg- Leu- Bug- pFPhe —NH2 (SEQ ID NO. 151) H- Ala- Ala- Lys- Arg- Arg- Leu- Ser- pFPhe —NH2 (SEQ ID NO. 152) H- Ala- Ala- Lys- Arg- Arg- Leu- Asp- pFPhe —NH2 (SEQ ID NO. 153) H- Ala- Ala- Lys- Arg- Arg- Leu- Asn- pFPhe —NH2 (SEQ ID NO. 154) H- Ala- Ala- Lys- Arg- Arg- Leu- pFPhe- Phe —NH2 (SEQ ID NO. 155) H- Ala- Ala- Lys- Arg- Arg- Leu- diClPhe Phe —NH2 (SEQ ID NO. 156) H- Ala- Ala- Lys- Arg- Arg- Leu- pClPhe- Phe —NH2 (SEQ ID NO. 157) H- Ala- Ala- Lys- Arg- Arg- Leu- mClPhe Phe —NH2 (SEQ ID NO. 158) H- Ala- Ala- Lys- Arg- Arg- Leu- oClPhe- Phe —NH2 (SEQ ID NO. 159) H- Ala- Ala- Lys- Arg- Arg- Leu- pIPhe- Phe —NH2 (SEQ ID NO. 160) H- Ala- Ala- Lys- Arg- Arg- Leu- TyrMe- Phe —NH2 (SEQ ID NO. 161) H- Ala- Ala- Lys- Arg- Arg- Leu- Thi- Phe —NH2 (SEQ ID NO. 162) H- Ala- Ala- Lys- Arg- Arg- Leu- Pya- Phe —NH2 (SEQ ID NO. 163) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- diClPhe —NH2 (SEQ ID NO. 164) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- pClPhe —NH2 (SEQ ID NO. 165) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- mClPhe —NH2 (SEQ ID NO. 166) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- oClPhe —NH2 (SEQ ID NO. 167) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- Phg —NH2 (SEQ ID NO. 168) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- TyrMe —NH2 (SEQ ID NO. 169) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- Thi —NH2 (SEQ ID NO. 170) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- Pya —NH2 (SEQ ID NO. 171) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- Inc —NH2 (SEQ ID NO. 172)

[0043] and the cyclic peptides: 5,8-cyclo-[H-His-Ala-Lys-Arg-Lys-Leu-Phe-Gly] (SEQ ID NO. 173) 5,8-cyclo-[H-His-Ala-Lys-Arg-Orn-Leu-Phe-Gly] (SEQ ID NO. 174)

[0044] In another aspect, the invention pertains to a peptide of the formula III or IV; H′X₂K′R₁R₂L′X₅F (formula III) (SEQ ID No. 175) or H′X₂K″R₁R₂L′FX₅ (formula IV) (SEQ ID No. 176) or a variant thereof, wherein:

[0045] H′ is nothing, His, D-His, Ala, Thi, Hse, Phe, or Dab;

[0046] X₂ is Ala, Ser, Abu, Val;

[0047] K′ is Lys, Arg, or Abu;

[0048] R₁ is Arg, Lys, or Gln; and

[0049] R₂ is Arg, forms a cyclic peptide with the C-terminal residue, Ser, or Cit;

[0050] L′ is Leu or Ile;

[0051] X₅ is Ile, Leu, Gly, or Ala; and

[0052] F′ is Phe, para-fluoroPhe, meta-fluoroPhe, L-Psa, 2-Nap,Dhp, or D-Psa.

[0053] In one embodiment, X₂ is alanine.

[0054] In one embodiment, X₅ isoleucine.

[0055] In another embodiment, the invention pertains to a peptide of the formula IV H′X₂K′R₁R₂L′F′X₅ (SEQ ID No. 176).

[0056] In another embodiment, the peptide is in a cyclic form by virtue of a linkage between the C-terminal residue and the residue 3 upstream to it.

[0057] In another embodiment, X₂ is Ala and X₅ is Ile.

[0058] In yet another embodiment, F′ is para-fluoro-Phe and H′ is Ala or nothing.

[0059] In another embodiment, K′ is Abu; R₁ is Gln; R₂ is Cit or Ser; and X₅ is Ala.

[0060] In still another embodiment, a peptide is selected from the group consisting of: H- his- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 36) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 47) H- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 48) H- Thi- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 50) H- Hse- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 51) H- Phe- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 52) H- Dab- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 53) H- His- Abu- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 55) H- His- Val- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 58) H- His- Ala- Arg- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 66) H- His- Ala- Lys- Arg- Arg- Ile- Ile- Phe —NH2 (SEQ ID NO. 83) H- His- Ala- Lys- Arg- Arg- Leu- Leu- Phe —NH2 (SEQ ID NO. 91) H- His- Ala- Lys- Arg- Arg- Leu- Ile- pFPhe —NH2 (SEQ ID NO. 103) H- His- Ala- Lys- Arg- Arg- Leu- Ile- 2Nap —NH2 (SEQ ID NO. 107) H- His Ala Lys Arg Arg Leu Ile D-Psa OH (SEQ ID NO. 115) H- His Ser Lys Arg Arg Leu Ile Dhp OH (SEQ ID NO. 119) H- Ala- Ala- Abu- Arg- Arg- Leu- Ile- pFPhe —NH2 (SEQ ID NO. 122) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- pFPhe —NH2 (SEQ ID NO. 123) H- Ala- Ala- Lys- Arg- Cit- Leu- Ile- pFPhe —NH2 (SEQ ID NO. 124) H- Ala- Ala- Lys- Arg- Arg- Leu- Ala- pFPhe —NH2 (SEQ ID NO. 125) H- Ala- Ala- Abu- Arg- Ser- Leu- Ile- pFPhe —NH2 (SEQ ID NO. 126) H- Ala- Ala- Lys- Gln- Arg- Leu- Ile- pFPhe —NH2 (SEQ ID NO. 127) H- Ala- Lys- Arg- Arg- Leu- Ile- pFPhe —NH2 (SEQ ID NO. 177)

[0061] In another aspect, the invention pertains to an assay for identifying candidate substances capable of binding to a cyclin associated with a G1 control CDK enzyme and/or inhibition of said enzyme, comprising; (a) bringing into contact i) a p21 derived peptide as defined in claim 1, ii) said cyclin or portion thereof or cyclin groove, iii) said CDK or portion thereof and iv) said candidate substance, under conditions wherein, in the absence of the candidate substance being an inhibitor of the cyclin/CDK interaction, the p21 derived peptide would bind to said cyclin or portion thereof or cyclin groove, and (b) monitoring any change in the expected binding of the p21 derived peptide and the cyclin or portion thereof or cyclin groove.

[0062] In yet another aspect, the invention pertains to an assay for the identification of compounds that interact with a cyclin or a cyclin when complexed with the physiologically relevant CDK, comprising; (a) incubating a candidate compound and peptide of formula I: X₁X₂X₃RX₄LX₅F (formula II) (SEQ ID No. 2) wherein X₁, X₃, X₄ and X₅ may be any amino acid and X₂ is serine or alanine; and variants thereof or a peptide of the formula III or IV: H′X′₂K′R₁R₂L′X′₅F′ (formula III) (SEQ ID No. 175) or H′X′₂K′R₁R₂L° F′X′₅ (formula IV) (SEQ ID No. 176) or a variant thereof, wherein H′ is His, nothing, D-His, Ala, Thi, Hse, Phe, or Dab; X′₂ is Ala, Ser, Abu, Val; K′ is Lys, Arg, or Abu; R₁ is Arg, Lys, or Gln; and R₂ is Arg, forms a cyclic peptide with the C-terminal residue, Ser, or Cit; L′ is Leu or Ile; X′₅ is Ile, Leu, Gly, or Ala; F′ is Phe, para-fluoroPhe, meta-fluoroPhe, L-Psa, 2-Nap,Dhp, or D-Psa and a cyclin or cyclin/CDK complex; (b) detecting binding of either the candidate compound or the peptide of formula II or III with cyclin.

[0063] In another aspect, the invention pertains to an assay for candidate compounds that interact with a cyclin by virtue of forming associations with at least two of the amino acids corresponding to the cyclin A amino acids L253, 1206 and R211.

[0064] In yet another aspect of the invention, the candidate compound additionally forms associations with at least one of the amino acids corresponding to the cyclin A amino acids E223, E224, D284, D283, L253, 1206 and R211.

[0065] In one embodiment, the candidate additionally forms associations with at least one of the amino acids corresponding to the cyclin A amino acids W217, V219, V221, S408, E411, Y225, I213, L214, G257, R250, Q254, T207 and L214.

[0066] In still another aspect, the candidate compound additionally forms associations with at least one of the amino acids corresponding to the cyclin A amino acids G222, Y225, I281, E223, E220, V279, A212, V215, L218, Q406, S408, M210, L253, L218, I239, V256 and M200.

[0067] In one embodiment, the cyclin is selected from cyclin A, cyclin E or cyclin D.

[0068] In another embodiment, the cyclin is cyclin A.

[0069] In one embodiment, the assay comprises use of a three dimensional model of a cyclin and a candidate compound.

[0070] In another embodiment, at least one of the assay components is bound to a solid phase.

[0071] In still another embodiment, the p21 derived peptide is labeled such as to emit a signal when bound to said cyclin.

[0072] In another embodiment, the cyclin is labeled such as to emit a signal when bound to the p21 derived peptide.

[0073] In one embodiment, one of the assay components is labeled with a fluorescence emitter and the signal is detected using fluorescence polarization techniques.

[0074] In another aspect, the invention pertains to a method of using a cyclin in a drug screening assay comprising: (a)selecting a candidate compound by performing rational drug design with a three-dimensional model of said cyclin, wherein said selecting is performed in conjunction with computer modeling; (b)contacting the candidate compound with the cyclin; and (c)detecting the binding affinity of the candidate compound for the cyclin groove; wherein a potential drug is selected on the basis of its having a greater affinity for the cyclin groove than that of a peptide of formula II: X₁X₂X₃RX₄LX₅F (formula II) (SEQ ID No. 2) wherein X₁, X₃, X₄ and X₅ may be any amino acid and X₂ is serine or alanine; and variants thereof or a peptide of formula III or IV: H′X′₂K′R₁R₂L′X′₅F′ (formula III) (SEQ ID No. 175) or H′X′₂K′R₁R₂L′F′X′₅ (formula IV) (SEQ ID No. 176) or a variant thereof, wherein H′ is His, nothing, D-His, Ala, Thi, Hse, Phe, or Dab; X′₂ is Ala, Ser, Abu, Val; K′ is Lys, Arg, or Abu; R₁ is Arg, Lys, or Gln; and R₂ is Arg, forms a cyclic peptide with the C-terminal residue, Ser, or Cit; L′ is Leu or Ile; X′₅ is Ile, Leu, Gly, or Ala; F′ is Phe, para-fluoroPhe, meta-fluoroPhe, L-Psa, 2-Nap,Dhp, or D-Psa.

[0075] In another aspect, the invention pertains to a method of using a cyclin in a drug screening assay comprising: (a)selecting a candidate compound by performing rational drug design with a three-dimensional model of said cyclin, wherein said selecting is performed in conjunction with computer modeling; (b)contacting the candidate compound with the cyclin; and (c)detecting whether said the candidate compound forms associations with at least the amino acids corresponding to the cyclin A amino acids L253, I206 and R211.

[0076] In one embodiment, the method further comprises detection of whether the candidate compound additionally forms associations with at least one of the amino acids corresponding to the cyclin A amino acids E223, E224, D284, D283, L253, I206 and R211.

[0077] In another embodiment, the method further comprises detection of whether the candidate compound additionally forms associations with at least one of the amino acids corresponding to the cyclin A amino acids W217, V219, V221, S408, E411, Y225, I213, L214, G257, R250, Q254, T207 and L214.

[0078] In another embodiment, the method further comprises detection of whether the candidate compound additionally forms associations with at least one of the amino acids corresponding to the cyclin A amino acids G222, Y225, I281, E223, E220, V279, A212, V215, L218, Q406, S408, M210, L253, L218, I239, V256 and M200.

[0079] In another aspect, the invention pertains to an assay for identifying candidate substances capable of inhibiting CDK in a cell, comprising;

[0080] (a) contacting a cell comprising a cyclin or portion thereof or cyclin groove, and a CDK or portion thereof, with a candidate substance under conditions where, in the absence of the candidate substance, the cyclin or portion thereof or cyclin groove and CDK or portion thereof would interact, and (b) monitoring any change in the activity of the CDK or portion thereof, wherein inhibition of CDK activity is indicated by one or more of: G0 and/o G1/S cell cycle arrest; cell cycle-related apoptosis; suppression of E2F transcription factor activity; hypophosphorylation of cellular pRb; and in vitro anti-proliferative effects.

[0081] In still another aspect, the invention pertains to use of a peptide in the preparation of a medicament for use in (a) inhibition of CDK2 or (b) in the treatment of proliferative disorders such as cancers and leukaemias where inhibition of CDK2 would be beneficial.

BRIEF DESCRIPTION OF THE DRAWINGS

[0082]FIG. 1 shows the effect of p21 (149-160) on CDK2-Cyclin E induced phopshorylation of different concentrations Histone 1. Yellow line (+marks)—1 mg/ml Histone 1, purple line (diamonds), 0.7 mg/ml Histone 1, blue line (×marks)—0.25 mg/ml Histone 1 and brown line (closed circles)—0.1 mg/ml Histone 1.

[0083]FIG. 2 shows that p21 (141-160)153A is a strong inhibitor of GST-Rb phopshorylation but not of Histone 1 phosphorylation induced by CDK2-Cyclin E kinase complex.

[0084]FIG. 3(a) shows interactions of p27(27 Ser-Ala-Cys-Arg-Asn-Leu-Phe-Gly³⁴ (SEQ ID NO. 178) segment with cyclin A groove (Russo, A. A.; Jeffrey, P. D.; Patten, A. K.; Massague, J.; Pavletich, N. P. Nature 1996, 382, 325-31). Panel B shows conformation of the same segment (top) compared with modelled cyclic Ser-Ala-(Lys-Arg-Lys-Leu-Phe-Gly (SEQ ID NO. 179) peptide (bottom).

[0085]FIG. 4 shows the 3-D structure of the peptide H-His-Ala-Lys-Arg-Arg-Leu-Ile-Phe-NH₂ (SEQ ID NO. 36)/cyclin A complex was generated using molecular docking techniques. The peptide structure is represented in black, while only the residues of the cyclin groove that make intermolecular contacts with the peptide are shown. The backbone of cyclin A is represented by the grey ribbon.

[0086]FIG. 5 shows comparison of the conformation of cyclin A-complexed structures of the p21- and p27-derived peptides H-His-Ala-Lys-Arg-Arg-Leu-Ile-Phe (SEQ ID NO. 36) and H-Ser-Ala-Cys-Arg-Asn-Leu-Phe-Gly-NH₂ (SEQ ID NO. 180). The positioning of the Leu and Phe side chains of the Leu-Ile-Phe and Leu-Phe-Gly motifs in the groove is remarkably similar, despite the different sequence order of these residues.

[0087]FIG. 6 shows comparison of modelled cyclin A groove-bound conformations of the p21(152-159)Ser153Ala peptides containing either Phe¹⁵⁹ (top) or pFPhe¹⁵⁹ (bottom).

DETAILED DESCRIPTION OF THE INVENTION

[0088] Although the peptides of the first aspect and in some embodiments of the second aspect, include the described CDK4-inhibitory motif RRLIF, the peptides of the present invention have been shown to display preferential selectivity for CDK2 over CDK4 in contrast to those described in Ball et al.(supra) who concluded that such p21 carboxy-terminal peptides “do not have high specific activity for CDK2 inhibition, they are potent inhibitors of CDK4 activity”. Thus, Ball et al. do not focus upon this region for further development for preferential CDK2 inhibitors, indeed p21₁₄₁₋₁₆₀ was shown by these authors to be 40 times more active against cyclinD1/CDK4 than cyclinE/CDK2. Thus, further surprising advantages of the above peptides relate to their specificity particularly for G1 control CDK's, such as CDK2/cyclinE and CDK2/cyclin A, as opposed to mitotic control enzymes including CDK's such as CDK1/cyclin B or A and protein kinase Cα (PKCα).

[0089] Further evidence of the unexpected observation that these peptides display activity against CDK4 and CDK2 is that Ball et al. described how N-terminal truncation of p21₁₄₁₋₁₆₀ reduced CDK4/cyclin D1 inhibitory activity. The disclosure therein of RRLIF (SEQ ID No. 5) as being the CDK4-inhibitory motif was made on a theoretical basis rather than a demonstration that a peptide of that size would retain inhibitory activity. Furthermore, of the prior art disclosures discussed above, only two 8-mer peptides have been shown to be active against cyclin A/CDK2, these being the E2F1 derived peptides PVKRRLFG (SEQ ID No. 8) and PVKRRLDL (SEQ ID No. 7). Thus, the present invention has demonstrated, in contrast to the information available in the art, that shorter, in some cases more specific and/or potent inhibitors of cyclin-CDK, especially cyclin E/CDK2 and cyclin A/CDK2 interaction may derived from within the sequence p21₁₄₁₋₁₆₀.

[0090] In one embodiment of the first aspect of the invention, the peptide may include a further amino acid residue at either the N- or C-terminus. The further residue is preferably selected from the polar residues C, N, Q, S, T and Y, and is preferably threonine when added to the N-terminus and serine, when added to the C-terminus. These last recited preferred embodiments correspond to the sequences 148-159 and 149-160 of p21 respectively. In an alternative embodiment, up to 7 amino acid residues may be deleted from the N-terminal end of formula I. Such truncation may therefore give rise to peptides corresponding to p21(150-159), p21(151-159), p21(152-159), p21(153-159), p21(154-159) p21(155-159) and p21(156-159) or wherein an additional serine residue is added to the C-terminal end to p21(150-160), p21(151-160), p21(152-160), p21(153-160), p21(154-160), p21(155-160) and p21(156-160). Preferably, from 2 to 7 residues are deleted, most preferably seven are deleted. In each of these preferred embodiments it is preferable that, when present the serine residue corresponding to p21(153) is replaced by an alanine residue.

[0091] Considering the second aspect of the invention, peptides and variants of the formula X₁X₂X₃RX₄LX₅F (SEQ ID No. 2) include peptides where one or more of:

[0092] (a) X1 may be deleted or may be any amino acid,

[0093] (b) X2 may be serine or alanine or a straight or branched chain amino acid,

[0094] (c) X3 may be a basic amino acid or straight or branched chain aliphatic amino acid,

[0095] (d) R may be unchanged or conservatively substituted (by basic amino acids),

[0096] (e) X4 may be any amino acid that is capable of providing at least one site for participating in hydrogen bonding,

[0097] (f) L may be unchanged or conservatively substituted,

[0098] (g) X5 may be any amino acid, or

[0099] (h) F may be unchanged or substituted by any aromatic amino acid.

[0100] More particularly, X₂ is preferably alanine as this provides a significant increase in the efficacy of the peptide and X₅ is preferably a non-polar amino acid residue, more preferably isoleucine or glycine, most preferably isoleucine. Of the remaining groups, X₁, X₃ and X₄, X₁ and X₄ are both preferably basic amino acid residue, X₁ is more preferably histidine and X₄ more preferably arginine. X₃ may be a basic or polar residue, preferably lysine or cysteine. A preferred peptide in accordance with the second aspect is that of SEQ ID No.3;

HX₂KRRLX₅F  (SEQ ID No. 3)

[0101] wherein X₂ and X₅ have the same meanings and preferences as above. When X₂ is serine and X₅ isoleucine the peptide corresponds to the sequence 152-159 of p21 and may hereinafter be referred to as p21(152-159). A further aspect of the invention therefore relates to a peptide HX₂KRRLX₅F (SEQ ID No. 3) and variants thereof, especially, wherein at least one amino acid residue is replaced by an alternative natural or unnatural replacement amino acid residue.

[0102] As used herein the term “variant” is used to include the peptides of SEQ ID Nos 1, 2 and 3 being modified by at least one of; deletion, addition or substitution of one or more amino acid residues, or by substitution of one or more natural amino acid residues by the corresponding D-stereomer or by a non-natural amino acid residue, chemical derivatives of the peptides, cyclic peptides derived from the peptides or from the peptide derivatives, dual peptides, multimers of the peptides and any of said peptides in the D-stereoisomer form or the order of the final two residues at the C-terminus residues are reversed; provided that such variants retain the activity of the parent peptide. As used hereinafter, the term “substitution” is used as to mean “replacement” i.e. substitution of an amino acid residue means its replacement e.g. with a different natural or non-natural amino acid residue.

[0103] Preferably, the variants involve the replacement of an amino acid residue by one or more, preferably one, of those selected from the residues of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0104] Such variants may arise from homologous substitution i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc. Non-homologous substitution may also occur i.e. from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine, diaminobutyric acid, norleucine, pyriylalanine, thienylalanine, naphthylalanine and phenylglycine.

[0105] As used herein, amino acids are classified according to the following classes;

[0106] basic; H, K, R

[0107] acidic; D, E

[0108] non-polar; A, F, G, I, L, M, P, V, W

[0109] polar; C, N, Q, S, T, Y,

[0110] (using the internationally accepted amino acid single letter codes)

[0111] and homologous and non-homologous substitution is defined using these classes. Thus, homologous substitution is used to refer to substitution from within the same class, whereas non-homologous substitution refers to substitution from a different class or by an unnatural amino acid.

[0112] The variants may also arise from replacement of an amino acid residue by an unnatural amino acid residue that may be homologous or non-homologous with that it is replacing. Such unnatural amino acid residues may be selected from;-alpha* and alpha-disubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halide derivatives of natural amino acids such as trifluorotyrosine*, β-Cl-phenylalanine*, p-Br-phenylalanine*, p-I-phenylalanine*, L-allyl-glycine*, β-alanine*, L-α-amino butyric acid*, L-γ-amino butyric acid*, L-α-amino isobutyric acid*, L-ε-amino caproic acid#, 7-amino heptanoic acid*, L-methionine sulfone#*, L-norleucine*, L-norvaline*, p-nitro-L-phenylalanine*, L-hydroxyproline#, L-thioproline*, methyl derivatives of phenylalanine (Phe) such as 4-methyl-Phe*, pentamethyl-Phe*, L-Phe (4-amino)#, L-Tyr (methyl)*, L-Phe (4-isopropyl)*, L-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxyl acid)*, L-diaminopropionic acid 4 and L-Phe (4-benzyl)*. The notation* has been utilised for the purpose of the discussion above, to indicate the hydrophobic nature of the derivative whereas # has been utilised to indicate the hydrophilic nature of the derivative, #* indicates amphipathic characteristics. The structures and accepted three letter codes of some of these and other unnatural amino acids are given in the Examples section.

[0113] With particular reference to the first aspect of the invention (SEQ ID No. 1), a variant peptide may involve the replacement of an amino acid residue by an alanine residue. In the first aspect of the present invention, such substitution preferably takes place at any of positions 150, 151, 152, 153, 154, 158 or 160 which all display a greater selectivity for CDK2/cyclin F inhibition than CDK4/cyclin D1 inhibition as described below. Most preferably, such alanine replacement occurs at position 153 where in addition to an increase in selectivity, the observed IC₅₀ is at least two orders of magnitude greater that for the corresponding parent peptide (p21₁₄₉₋₁₆₀). In respect of the second aspect of the invention, it is also preferable that amino acid replacement is by an alanine residue, most preferably at the 153 position (X₂). Furthermore, in respect of this aspect of the invention, the variant may include the deletion of the N-terminal asparagine residue resulting in the peptide corresponding to p21(150-159). According the first aspect, a preferable peptide is one including a serine residue at the C-terminus such as the peptide D F Y H A K R R L I F S (SEQ ID No. 19)

[0114] As discussed above, variants also include inversion of the two C-terminal amino acid residues and cyclic peptides, both of which are preferred independently as well as when taken together or in combination with any other variant. When such a variant is applied to the second or third aspects of the invention, it is to the exclusion of the peptide PVKRRLFG (SEQ ID No. 8), unless in cyclic form.

[0115] With regard to cyclic peptides, these are preferably formed by linkage between the C-terminal amino acid residue and any upstream amino acid residue, preferably 3 amino acid residues upstream to it. Those skilled in the art will be aware as to the nature of such cyclic linkages. In some instances the participating amino acids may require modification in order to facilitate such linkage. In the context of the present invention, cyclic peptides are most conveniently prepared using variants wherein the two C-terminal amino acids are reversed, I and F when considering the first aspect of the invention, X₅ and the terminal phenylalanine residue in the second aspect etc. resulting in a linkage between I or X₅ and an upstream residue. In such circumstances the terminal amino acid residue (I or X₅) is preferably modified to be glycine, the upstream amino acid residue preferably being modified to be lysine or ornithine.

[0116] Thus, in accordance with the first aspect of the invention, the peptide may be selected from: D F Y H A K R R L I F S, (SEQ ID No. 19) T D F Y H S K R R L I F, (SEQ ID No. 14) A F Y H S K R R L I F S, (SEQ ID No. 15) D A Y H S K R R L I F S, (SEQ ID No. 16) D F A H S K R R L I F S, (SEQ ID No. 17) D F Y A S K R R L I F S, (SEQ ID No. 18) D F Y H A K R R L I F S, (SEQ ID No. 19) D F Y H S A R R L I F S, (SEQ ID No. 20) D F Y H S K R A L I F S, (SEQ ID No. 21) D F Y H S K R R L A F S, (SEQ ID No. 22) D F Y H S K R R L I F A, (SEQ ID No. 23) F Y H S K R R L I F S, (SEQ ID No. 24) Y H S K R R L I F S, (SEQ ID No. 25) H S K R R L I F S, (SEQ ID No. 26) D F Y H S K R R L I F, (SEQ ID No. 1) F Y H S K R R L I F, (SEQ ID No. 27) Y H S K R R L I F, (SEQ ID No. 28) H S K R R L I F, (SEQ ID No. 29) S K R R L I F, (SEQ ID No. 30) K R R L I F, (SEQ ID No. 31) H- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 32) H- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 33) H- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 34) H- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 35) H- His- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID NO. 36) H- Asn- Leu- Phe- Gly —NH2 (SEQ ID NO. 37) H- Arg- Asn- Leu- Phe- Gly —NH2 (SEQ ID NO. 38) H- Abu- Arg- Asn- Leu- Phe- Gly —NH2 (SEQ ID NO. 39) H- Ala- Abu- Arg- Asn- Leu- Phe- Gly —NH2 and (SEQ ID NO. 40) H- Ser- Ala- Abu- Arg- Asn- Leu- Phe- Gly —NH2 (SEQ ID NO. 41)

[0117] Considering X₁X₂X₃RX₄LX₅F (SEQ ID No. 2), preferred peptides and variants thereof may include any one of or optionally at least one or more of the following;

[0118] (a) X₁ is histidine, deleted or replaced by a natural or unnatural amino acid residue-such as alanine, 3-pyridylalanine (Pya), 2-thienylalanine (Thi), homoserine (Hse), phenylalanine, or diaminobutyric acid (Dab),

[0119] (b) X₂ is alanine or an alternative natural or unnatural amino acid residue having a smaller or slightly larger aromatic or aliphatic side chain, such as glycine, aminobutyric acid (Abu), norvaline (Nva), t-butylglycine(Bug), valine, isoleucine, phenylglycine (Phg) or phenylalanine,

[0120] (c) X₃ is lysine or either a basic residue such as arginine or an uncharged natural or unnatural amino acid residue, such as norleucine (Nle), aminobutyric acid (Abu) or leucine,

[0121] (d) arginine is replaced by either a basic residue such as lysine or an uncharged natural or unnatural amino acid residue, such as citrulline (Cit), homoserine, histidine, norleucine (Nle) or glutamine,

[0122] (e) X₄ is or a natural or unnatural amino acid residue, such as asparagine, proline, serine, aminoisobutyric acid (Aib) or sarcosine (Sar), or an amino acid residue capable of forming a cyclic linkage such as lysine or ornithine,

[0123] (f) leucine is replaced with a natural or unnatural amino acid residue having a slightly larger aromatic or aliphatic side chain, such as norleucine, norvaline, cyclohexylalanine (Cha), phenylalanine or 1-naphthylalanine (1Nal),

[0124] (g) X₅ is isoleucine or an alternative natural or unnatural amino acid residue having a slightly larger aromatic or aliphatic side chain, such as norleucine, norvaline, cyclohexylalanine (Cha), phenylalanine or 1-naphthylalanine (1Nal),

[0125] (h) phenylalanine is replaced with a natural or unnatural amino acid such as leucine, cyclohexylalanine (Cha), homophenylalanine (Hof), tyrosine, para-fluorophenylalanine (pFPhe), meta-fluorophenylalanine (mFPhe), trptophan, 1-naphthylalanine (1Nal), 2naphthylalanine (2Nal), biphenylalanine(Bip) or (Tic),

[0126] (j) X₅ and the terminal phenylalanine residue are reversed, or

[0127] (j) the peptide is in cyclic form by for example, the formation of a linkage between the side chain of X₄ and the C-terminus residue.

[0128] In accordance with the second embodiment of the invention, the peptide may be selected from; H S K R R L I F, (SEQ ID No. 29) H A K R R L I F, (SEQ ID No. 42) H S K R R L F G, (SEQ ID No. 43) H A K R R L F G, (SEQ ID No. 44) K A C R R L F G, (SEQ ID No. 45) K A C R R L I F, (SEQ ID No. 46) X1 X2 X3 R X4 L X5 F (SEQ ID No. 2) H- His- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 36) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 47) H- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 48) H- Pya- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 49) H- Thi- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 50) H- Hse- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 51) H- Phe- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 52) H- Dab- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 53) H- His- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 36) H- His- Gly- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 54) H- His- Abu- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 55) H- His- Nva- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 56) H- His- Bug- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 57) H- His- Val- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 58) H- His- Ile- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 59) H- His- Phg- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 60) H- His- Phe- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 61) H- His- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 36) H- His- Ala- Ala- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 62) H- His- Ala- Nle- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 63) H- His- Ala- Abu- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 64) H- His- Ala- Leu- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 65) H- His- Ala- Arg- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 66) H- His- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 36) H- His- Ala- Lys- Ala- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 67) H- His- Ala- Lys- Cit- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 68) H- His- Ala- Lys- Hse- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 69 H- His- Ala- Lys- His- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 70) H- His- Ala- Lys- Nle- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 71) H- His- Ala- Lys- Gln- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 72) H- His- Ala- Lys- Lys- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 73) H- His- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 36) H- His- Ala- Lys- Arg- Ala- Leu- Ile- Phe —NH2 (SEQ ID No. 74) H- His- Ala- Lys- Arg- Asn- Leu- Ile- Phe —NH2 (SEQ ID No. 75) H- His- Ala- Lys- Arg- Pro- Leu- Ile- Phe —NH2 (SEQ ID No. 76) H- His- Ala- Lys- Arg- Ser- Leu- Ile- Phe —NH2 (SEQ ID No. 77) H- His- Ala- Lys- Arg- Aib- Leu- Ile- Phe —NH2 (SEQ ID No. 78) H- His- Ala- Lys- Arg- Sar- Leu- Ile- Phe —NH2 (SEQ ID No. 79) H- His- Ala- Lys- Arg- Cit- Leu- Ile- Phe —NH2 (SEQ ID No. 80) H- His- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 36) H- His- Ala- Lys- Arg- Arg- Ala- Ile- Phe —NH2 (SEQ ID No. 81) H- His- Ala- Lys- Arg- Arg- leu Ile- Phe —NH2 (SEQ ID No. 82) H- His- Ala- Lys- Arg- Arg- Ile- Ile- Phe —NH2 (SEQ ID No. 83) H- His- Ala- Lys- Arg- Arg- Val- Ile- Phe —NH2 (SEQ ID No. 84) H- His- Ala- Lys- Arg- Arg- Nle- Ile- Phe —NH2 (SEQ ID No. 85) H- His- Ala- Lys- Arg- Arg- Nva- Ile- Phe —NH2 (SEQ ID No. 86) H- His- Ala- Lys- Arg- Arg- Cha- Ile- Phe —NH2 (SEQ ID No. 87) H- His- Ala- Lys- Arg- Arg- Phe- Ile- Phe —NH2 (SEQ ID No. 88) H- His- Ala- Lys- Arg- Arg- 1Nap- Ile- Phe —NH2 (SEQ ID No. 89) H- His- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 36) H- His- Ala- Lys- Arg- Arg- Leu- Ala- Phe —NH2 (SEQ ID No. 90) H- His- Ala- Lys- Arg- Arg- Leu- Leu- Phe —NH2 (SEQ ID No. 91) H- His- Ala- Lys- Arg- Arg- Leu- Val- Phe —NH2 (SEQ ID No. 92) H- His- Ala- Lys- Arg- Arg- Leu- Nle- Phe —NH2 (SEQ ID No. 93) H- His- Ala- Lys- Arg- Arg- Leu- Nva- Phe —NH2 (SEQ ID No. 94) H- His- Ala- Lys- Arg- Arg- Leu- Cha- Phe —NH2 (SEQ ID No. 95) H- His- Ala- Lys- Arg- Arg- Leu- Phe- Phe —NH2 (SEQ ID No. 96) H- His- Ala- Lys- Arg- Arg- Leu- 1Nap- Phe —NH2 (SEQ ID No. 97) H- His- Ala- Lys- Arg- Arg- Leu- Phe —NH2 (SEQ ID No. 98) H- His- Ala- Lys Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 36) H- His- Ala- Lys- Arg- Arg- Leu- Ile- Leu —NH2 (SEQ ID No. 99) H- His- Ala- Lys- Arg- Arg- Leu- Ile- Cha —NH2 (SEQ ID No. 100) H- His- Ala- Lys- Arg- Arg- Leu- Ile- Hof —NH2 (SEQ ID No. 101) H- His- Ala- Lys- Arg- Arg- Leu- Ile- Tyr —NH2 (SEQ ID No. 102) H- His- Ala- Lys- Arg- Arg- Leu- Ile- pFPhe —NH2 (SEQ ID No. 103) H- His- Ala- Lys- Arg- Arg- Leu- Ile- mFPhe —NH2 (SEQ ID No. 104) H- His- Ala- Lys- Arg- Arg- Leu- Ile- Trp —NH2 (SEQ ID No. 105) H- His- Ala- Lys- Arg- Arg- Leu- Ile- 1Nap —NH2 (SEQ ID No. 106) H- His- Ala- Lys- Arg- Arg- Leu- Ile- 2Nap —NH2 (SEQ ID No. 107) H- His- Ala- Lys- Arg- Arg- Leu- Ile- Lys —NH2 (SEQ ID No. 108) H- His- Ala- Lys- Arg- Arg- Leu- Ile- Tic —NH2 (SEQ ID No. 109) H- His- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 36) H- His Ala Lys Arg Arg Leu Ile L-Pse OH (SEQ ID No. 110) H- His Ala Lys Arg Arg Leu Ile D-Pse OH (SEQ ID No. 111) H- His Ser Lys Arg Arg Leu Ile L-Pse OH (SEQ ID No. 112) H- His Ser Lys Arg Arg Leu Ile D-Pse OH (SEQ ID No. 113) H- His Ala Lys Arg Arg Leu Ile L-Psa OH (SEQ ID No. 114) H- His Ala Lys Arg Arg Leu Ile D-Psa OH (SEQ ID No. 115) H- His Ser Lys Arg Arg Leu Ile L-Psa OH (SEQ ID No. 116) H- His Ser Lys Arg Arg Leu Ile D-Psa OH (SEQ ID No. 117) H- His Ala Lys Arg Arg Leu Ile Dhp OH (SEQ ID No. 118) H- His Ser Lys Arg Arg Leu Ile Dhp OH (SEQ ID No. 119) H- His Ala Lys Arg Arg Leu Ile Pheol (SEQ ID No. 120) H- His Ser Lys Arg Arg Leu Ile Pheol (SEQ ID No. 121) H- His- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 36) H- Ala- Ala- Abu- Arg- Arg- Leu- Ile- pFPhe —NH2 (SEQ ID No. 122) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- pFPhe —NH2 (SEQ ID No. 123) H- Ala- Ala- Lys- Arg- Cit- Leu- Ile- pFPhe —NH2 (SEQ ID No. 124) H- Ala- Ala- Lys- Arg- Arg- Leu- Ala- pFPhe —NH2 (SEQ ID No. 125) H- Ala- Ala- Abu- Arg- Ser- Leu- Ile- pFPhe —NH2 (SEQ ID No. 126) H- Ala- Ala- Lys- Gln- Arg- Leu- Ile- pFPhe —NH2 (SEQ ID No. 127) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- pFPhe —NH2 (SEQ ID No. 128) H- Gly- Ala- Lys- Arg- Arg- Leu- Ile- pFPhe —NH2 (SEQ ID No. 129) H- Ala- Ala- Lys- hArg- Arg- Leu- Ile- pFPhe —NH2 (SEQ ID No. 130) H- Ala- Ala- Lys- Ser- Arg- Leu- Ile- pFPhe —NH2 (SEQ ID No. 131) H- Ala- Ala- Lys- Hse- Arg- Leu- Ile- pFPhe —NH2 (SEQ ID No. 132) H- Ala- Ala- Lys- Arg- Lys- Leu- Ile- pFPhe —NH2 (SEQ ID No. 133) H- Ala- Ala- Lys- Arg- Orn- Leu- Ile- pFPhe —NH2 (SEQ ID No. 134) H- Ala- Ala- Lys- Arg- Gln- Leu- Ile- pFPhe —NH2 (SEQ ID No. 135) H- Ala- Ala- Lys- Arg- Hse- Leu- Ile- pFPhe —NH2 (SEQ ID No. 136) H- Ala- Ala- Lys- Arg- Thr- Leu- Ile- pFPhe —NH2 (SEQ ID No. 137) H- Ala- Ala- Lys- Arg- Nva- Leu- Ile- pFPhe —NH2 (SEQ ID No. 138) H- Ala- Ala- Lys- Arg- Arg- Phg- Ile- pFPhe —NH2 (SEQ ID No. 139) H- Ala- Ala- Lys- Arg- Arg- Met- Ile- pFPhe —NH2 (SEQ ID No. 140) H- Ala- Ala- Lys- Arg- Arg- Ala- Ile- pFPhe —NH2 (SEQ ID No. 141) H- Ala- Ala- Lys- Arg- Arg- Hof- Ile- pFPhe —NH2 (SEQ ID No. 142) H- Ala- Ala- Lys- Arg- Arg- hLeu- Ile- pFPhe —NH2 (SEQ ID No. 143) H- Ala- Ala- Lys- Arg- Arg- aIle- Ile- pFPhe —NH2 (SEQ ID No. 144) H- Ala- Ala- Lys- Arg- Arg- Leu- Gly- pFPhe —NH2 (SEQ ID No. 145) H- Ala- Ala- Lys- Arg- Arg- Leu- βAla pFPhe —NH2 (SEQ ID No. 146) H- Ala- Ala- Lys- Arg- Arg- Leu- Phg- pFPhe —NH2 (SEQ ID No. 147) H- Ala- Ala- Lys- Arg- Arg- Leu- Aib- pFPhe —NH2 (SEQ ID No. 148) H- Ala- Ala- Lys- Arg- Arg- Leu- Sar- pFPhe —NH2 (SEQ ID No. 149) H- Ala- Ala- Lys- Arg- Arg- Leu- Pro- pFPhe —NH2 (SEQ ID No. 150) H- Ala- Ala- Lys- Arg- Arg- Leu- Bug- pFPhe —NH2 (SEQ ID No. 151) H- Ala- Ala- Lys- Arg- Arg- Leu- Ser- pFPhe —NH2 (SEQ ID No. 152) H- Ala- Ala- Lys- Arg- Arg- Leu- Asp- pFPhe —NH2 (SEQ ID No. 153) H- Ala- Ala- Lys- Arg- Arg- Leu- Asn- pFPhe —NH2 (SEQ ID No. 154) H- Ala- Ala- Lys- Arg- Arg- Leu- pFPhe- Phe —NH2 (SEQ ID No. 155) H- Ala- Ala- Lys- Arg- Arg- Leu- diClPhe Phe —NH2 (SEQ ID No. 156) H- Ala- Ala- Lys- Arg- Arg- Leu- pClPhe- Phe —NH2 (SEQ ID No. 157) H- Ala- Ala- Lys- Arg- Arg- Leu- mClPhe Phe —NH2 (SEQ ID No. 158) H- Ala- Ala- Lys- Arg- Arg- Leu- oClPhe- Phe —NH2 (SEQ ID No. 159) H- Ala- Ala- Lys- Arg- Arg- Leu- plPhe- Phe —NH2 (SEQ ID No. 160) H- Ala- Ala- Lys- Arg- Arg- Leu- TyrMe- Phe —NH2 (SEQ ID No. 161) H- Ala- Ala- Lys- Arg- Arg- Leu- Thi- Phe —NH2 (SEQ ID No. 162) H- Ala- Ala- Lys- Arg- Arg- Leu- Pya- Phe —NH2 (SEQ ID No. 163) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- diClPhe —NH2 (SEQ ID No. 164) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- pClPhe —NH2 (SEQ ID No. 165) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- mClPhe —NH2 (SEQ ID No. 166) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- oClPhe —NH2 (SEQ ID No. 167) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- Phg —NH2 (SEQ ID No. 168) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- TyrMe —NH2 (SEQ ID No. 169) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- Thi —NH2 (SEQ ID No. 170) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- Pya —NH2 (SEQ ID No. 171) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- Inc —NH2 (SEQ ID No. 172)

[0129] and the cyclic peptides; 5,8-cyclo-[H-His-Ala-Lys-Arg-Lys-Leu-Phe-Gly] (SEQ ID No. 173) 5,8-cyclo-[H-His-Ala-Lys-Arg-Orn-Leu-Phe-Gly] (SEQ ID No. 174)

[0130] In another preferred embodiment, the invention relates to a peptide selected from the following: H Ala Ala Abu Arg Ser Leu Ile pFPhe NH₂ (SEQ ID No. 126) H Ala Ala Abu Arg Ser Leu Ile Gly NH₂ (SEQ ID No. 290) H Ala Ala Abu Arg Ser Leu mClPhe pFPhe NH₂ (SEQ ID No. 291) H Ala Ala Abu Arg Ser Leu mClPhe Gly NH₂ (SEQ ID No. 292)

[0131] With particular reference to SEQ ID No. 3, a variant peptide may additionally involve the replacement of an amino acid residue by an alanine residue, the deletion of X₁ or the reversal of X₅ and the terminal phenylalanine residue. These options are also applicable to the peptide SEQ ID No 3 which may therefore, by way of example result in the peptides X₂KRRLX₅F (SEQ ID No. 181) and Hx₂KRRLFX₅ (SEQ ID No. 182). Most preferably, the peptide is H A K R R L I F (SEQ ID No. 42). Further variants those discussed below.

[0132] More preferably with respect to H X₂ K R R L X₅F (SEQ ID No. 3) preferred peptides and variants thereof may include any one of or optionally at least one or more of the following;

[0133] (a) His is unchanged, deleted or replaced by D-His, Ala, Thi, Hse, Phe, or Dab,

[0134] (b) X₂ is Ala unchanged or replaced by Ser, Abu Bug or Val,

[0135] (c) Lys is unchanged or replaced by Arg or Abu,

[0136] (d) Arg is unchanged or replaced by Lys, Cit, or Gln,

[0137] (e) Arg is unchanged or modified to form a cyclic peptide with the C-terminal residue, or replaced by Cit or Ser,

[0138] (f) Leu is unchanged or replaced by Ile,

[0139] (g) X₅ is Ile unchanged, replaced by Leu or Gly if reversed with Phe,

[0140] (h) Phe is unchanged or replaced by para-fluoroPhe, meta-fluoroPhe, L-Psa, 2-Nap or Dhp,

[0141] (i) the two C-terminal residue are reversed, or

[0142] (j) the peptide is in cyclic form by virtue of a linkage between the C-terminal residue and the residue 3 upstream to it.

[0143] Especially preferred are peptides wherein X₂ is Ala and X₅ is Ile, incorporating more than one of the above variations particularly where Phe is replaced by para-fluoro-Phe and His is replaced by Ala or is deleted. Of such peptides, especially preferred are those that include further modifications where:

[0144] (a) Lys is replaced by Abu,

[0145] (b) the first Arg residue is replaced by Gln and

[0146] (c) the second Arg residue is replaced by Cit or Ser and,

[0147] (d) Ile is replaced by Ala.

[0148] Thus, preferred peptides in accordance with the preferred sequence H A K R R L I F (SEQ ID No. 42) include; His152 H- his- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 183) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 47) H- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 148) H- Thi- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 50) H- Hse- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 51) H- Phe- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 52) H- Dab- Ala- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 54) Ala153 H- His- Abu- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 55) H- His- Val- Lys- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 58) Lys154 H- His- Ala- Arg- Arg- Arg- Leu- Ile- Phe —NH2 (SEQ ID No. 66) Leu157 H- His- Ala- Lys- Arg- Arg- Ile- Ile- Phe —NH2 (SEQ ID No. 83) Ile158 H- His- Ala- Lys- Arg- Arg- Leu- Leu- Phe —NH2 (SEQ ID No. 91) Phe159 H- His- Ala- Lys- Arg- Arg- Leu- Ile- pFPhe —NH2 (SEQ ID No. 103) H- His- Ala- Lys- Arg- Arg- Leu- Ile- 2Nap —NH2 (SEQ ID No. 107) H- His Ala Lys Arg Arg Leu Ile D-Psa OH (SEQ ID No. 115) H- His Ser Lys Arg Arg Leu Ile Dhp OH (SEQ ID No. 119) Multiples H- Ala- Ala- Abu- Arg- Arg- Leu- Ile- pFPhe —NH2 (SEQ ID No. 122) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- pFPhe —NH2 (SEQ ID No. 123) H- Ala- Ala- Lys- Arg- Cit- Leu- Ile- pFPhe —NH2 (SEQ ID No. 124) H- Ala- Ala- Lys- Arg- Arg- Leu- Ala- pFPhe —NH2 (SEQ ID No. 125) H- Ala- Ala- Abu- Arg- Ser- Leu- Ile- pFPhe —NH2 (SEQ ID No. 126) H- Ala- Ala- Lys- Gln- Arg- Leu- Ile- pFPhe —NH2 (SEQ ID No. 127) H- Ala- Lys- Arg- Arg- Leu- Ile- pFPhe —NH2 (SEQ ID No. 177)

[0149] The three letter notations appearing above are in accordance with IUPAC convention. The structure of various unnatural amino acid derivatives are provided in the introduction to the Examples, further expansion on nomenclature being given above.

[0150] The peptides of the present invention may be subjected to a further modification that is beneficial in the context of the present invention being conversion of the free carboxyl group of the carboxy terminal amino acid residue, to a carboxamide group. By way of example, when the peptide is of SEQ ID No.1 the carboxy terminal phenylalanine residue may have its carboxyl group converted into a carboxamide group. This modification is believed to enhance the stability of the peptide. Thus, the C-terminal amino acid residue may be in the form —C(O)—NRR′, wherein R and R′ are each independently selected from hydrogen, C1-6 alkyl, C1-6 alkylene or C1-6 alkynyl (collectively referred to “alk”), aryl such as benzyl or alkaryl, each optionally substituted by heteroatoms such as O, S or N. Preferably at least one of R or R′ is hydrogen, most preferably, they are both hydrogen. Thus, the present invention therefore encompasses the peptides wherein the C-terminal amino acid residue is in the carboxyl or carboxamide form.

[0151] In one preferred embodiment, the invention relates to peptides of formula V

RX₆X₇X₈X₉  (formula V) (SEQ ID No. 293)

[0152] wherein

[0153] X₆ is arginine, serine or lysine;

[0154] X₇ is leucine, isoleucine or valine;

[0155] X₈ is asparagine, alanine, glycine or isoleucine; and

[0156] X₉ is phenylalanine;

[0157] or variants thereof.

[0158] More preferably, the invention relates to peptides of formula V (SEQ ID No. 293)or variants thereof wherein the peptide is modified by at least one of a deletion, addition or substitution of one or more amino acid residues, or by substitution of one or more natural amino acid residues by the corresponding D-stereomer or by a non-natural amino acid residue, chemical derivatives of the peptides, cyclic peptides derived from the peptides or from the peptide derivatives, dual peptides, multimers of the peptides and any of said peptides in the D-stereomer form, or the order of the final two residues at the C-terminal end are reversed.

[0159] Even more preferably still, the invention relates to peptides of formula V (SEQ ID No. 293), or variants thereof, wherein: (a) R is unchanged or conservatively substituted (by a basic amino acid), (b) X₆ is substituted by any amino acid capable of providing at least one site for participating in hydrogen bonding, (c) X₇ is unchanged or conservatively substituted, (d) X₈ is unchanged or conservatively substituted, (e) X₉ is unchanged or substituted by any aromatic amino acid.

[0160] In another preferred embodiment, the invention relates to peptides of formula V (SEQ ID No. 293), or variants thereof, wherein:

[0161] (a) R is replaced by either a basic residue such as lysine or an uncharged natural or unnatural amino acid residue, such as citrulline (Cit), homoserine, histidine, norleucine (Nle), or glutamine,

[0162] (b) X₆ is replaced by a natural or unnatural amino acid residue such as asparagine, proline, aminoisobutyric acid (Aib) or sarcosine (Sar), or an amino acid residue capable of forming a cyclic linkage such as ornithine,

[0163] (c) X₇ is replaced with a natural or unnatural amino acid residue having a slightly larger aromatic or aliphatic side chain, such as norleucine, norvaline, cyclohexylalanine (Cha), phenylalanine or 1-naphthylalanine (1 Nal),

[0164] (d) X₈ is replaced with a natural or unnatural amino acid residue having a slightly larger aromatic or aliphatic side chain, such as norleucine, norvaline, cyclohexylalanine (Cha), phenylalanine or 1-naphthylalanine (1 Nal),

[0165] (e) X₉ is replaced with a natural or unnatural amino acid such as leucine, cyclohexylalanine (Cha), homophenylalanine (Hof), tyrosine, para-fluorophenylalanine (pFPhe), meta-fluorophenylalanine (mFPhe), trptophan, 1-naphthylalanine (1Nal), 2-naphthylalanine (2Nal), meta-chlorophenylalanine (mClPhe),biphenylalanine(Bip) or (Tic).

[0166] In one preferred embodiment, the invention relates to peptides of formula V

RX₆X₇X₈X₉  (formula V) (SEQ ID No. 293)

[0167] wherein

[0168] X₆ is arginine, serine or lysine;

[0169] X₇ is leucine, isoleucine or valine;

[0170] X₈ is asparagine, alanine, glycine or isoleucine; and

[0171] X₉ is phenylalanine;

[0172] or variants thereof.

[0173] More preferably, the invention relates to peptides of formula V or variants thereof wherein the peptide is modified by at least one of a deletion, addition or substitution of one or more amino acid residues, or by substitution of one or more natural amino acid residues by the corresponding D-stereomer or by a non-natural amino acid residue, chemical derivatives of the peptides, cyclic peptides derived from the peptides or from the peptide derivatives, dual peptides, multimers of the peptides and any of said peptides in the D-stereomer form, or the order of the final two residues at the C-terminal end are reversed.

[0174] Even more preferably still, the invention relates to peptides of formula V, or variants thereof, wherein: (a) R is unchanged or conservatively substituted (by a basic amino acid), (b) X₆ is substituted by any amino acid capable of providing at least one site for participating in hydrogen bonding, (c) X₇ is unchanged or conservatively substituted, (d) X₈ is unchanged or conservatively substituted, (e) X₉ is unchanged or substituted by any aromatic amino acid.

[0175] In another preferred embodiment, the invention relates to peptides of formula V (SEQ ID No. 293), or variants thereof, wherein:

[0176] (a) R is replaced by either a basic residue such as lysine or an uncharged natural or unnatural amino acid residue, such as citrulline (Cit), homoserine, histidine, norleucine (Nle), or glutamine,

[0177] (d) X₆ is replaced by a natural or unnatural amino acid residue such as asparagine, proline, aminoisobutyric acid (Aib) or sarcosine (Sar), or an amino acid residue capable of forming a cyclic linkage such as omithine,

[0178] (e) X₇ is replaced with a natural or unnatural amino acid residue having a slightly larger aromatic or aliphatic side chain, such as norleucine, norvaline, cyclohexylalanine (Cha), phenylalanine or 1-naphthylalanine (1Nal),

[0179] (d) X₈ is replaced with a natural or unnatural amino acid residue having a slightly larger aromatic or aliphatic side chain, such as norleucine, norvaline, cyclohexylalanine (Cha), phenylalanine or 1-naphthylalanine (1Nal),

[0180] (e) X₉ is replaced with a natural or unnatural amino acid such as leucine, cyclohexylalanine (Cha), homophenylalanine (Hof), tyrosine, para-fluorophenylalanine (pFPhe), meta-fluorophenylalanine (mFPhe), trptophan, 1-naphthylalanine (1Nal), 2-naphthylalanine (2Nal), meta-chlorophenylalanine (mClPhe),biphenylalanine(Bip) or (Tic).

[0181] In one particularly preferred embodiment, the invention relates to peptides of formula V (SEQ ID No. 293), or variants thereof, wherein the N-terminal is acylated.

[0182] In another particularly preferred embodiment, the invention relates to peptides of formula. V, or variants thereof, wherein R is substituted by citrulline.

[0183] Even more preferably, the invention relates to peptides of formula V (SEQ ID No. 293), or variants thereof, which are selected from the following: H- Arg Arg Leu Asn Phe NH₂ (SEQ ID No. 294) H- Arg Arg Leu Asn pFF NH₂ (SEQ ID No. 295) H- Arg Arg Leu Asn mClF NH₂ (SEQ ID No. 296) H- Arg Arg Leu Ala Phe NH₂ (SEQ ID No. 297) H- Arg Arg Leu Ala pFF NH₂ (SEQ ID No. 298) H- Arg Arg Leu Ala mClF NH₂ (SEQ ID No. 299) H- Arg Arg Leu Gly Phe NH₂ (SEQ ID No. 300) H- Arg Arg Leu Gly pFF NH₂ (SEQ ID No. 301) H- Arg Arg Leu Gly mClF NH₂ (SEQ ID No. 302) H- Arg Arg Ile Asn Phe NH₂ (SEQ ID No. 303) H- Arg Arg Ile Asn pFF NH₂ (SEQ ID No. 304) H- Arg Arg Ile Asn mClF NH₂ (SEQ ID No. 305) H- Arg Arg Ile Ala Phe NH₂ (SEQ ID No. 306) H- Arg Arg Ile Ala pFF NH₂ (SEQ ID No. 307) H- Arg Arg Ile Ala mClF NH₂ (SEQ ID No. 308) H- Arg Arg Ile Gly Phe NH₂ (SEQ ID No. 309) H- Arg Arg Ile Gly pFF NH₂ (SEQ ID No. 310) H- Arg Arg Ile Gly mClF NH₂ (SEQ ID No. 311) H- Arg Arg Val Asn Phe NH₂ (SEQ ID No. 312) H- Arg Arg Val Asn pFF NH₂ (SEQ ID No. 313) H- Arg Arg Val Asn mClF NH₂ (SEQ ID No. 314) H- Arg Arg Val Ala Phe NH₂ (SEQ ID No. 315) H- Arg Arg Val Ala pFF NH₂ (SEQ ID No. 316) H- Arg Arg Val Ala mClF NH₂ (SEQ ID No. 317) H- Arg Arg Val Gly Phe NH₂ (SEQ ID No. 318) H- Arg Arg Val Gly pFF NH₂ (SEQ ID No. 319) H- Arg Arg Val Gly mClF NH₂ (SEQ ID No. 320) H- Arg Ser Leu Asn Phe NH₂ (SEQ ID No. 321) H- Arg Ser Leu Asn pFF NH₂ (SEQ ID No. 322) H- Arg Ser Leu Asn mClF NH₂ (SEQ ID No. 323) H- Arg Ser Leu Ala Phe NH₂ (SEQ ID No. 324) H- Arg Ser Leu Ala pFF NH₂ (SEQ ID No. 325) H- Arg Ser Leu Ala mClF NH₂ (SEQ ID No. 326) H- Arg Ser Leu Gly Phe NH₂ (SEQ ID No. 327) H- Arg Ser Leu Gly pFF NH₂ (SEQ ID No. 328) H- Arg Ser Leu Gly mClF NH₂ (SEQ ID No. 329) H- Arg Ser Ile Asn Phe NH₂ (SEQ ID No. 330) H- Arg Ser Ile Asn pFF NH₂ (SEQ ID No. 331) H- Arg Ser Ile Asn mClF NH₂ (SEQ ID No. 332) H- Arg Ser Ile Ala Phe NH₂ (SEQ ID No. 333) H- Arg Ser Ile Ala pFF NH₂ (SEQ ID No. 334) H- Arg Ser Ile Ala mClF NH₂ (SEQ ID No. 335) H- Arg Ser Ile Gly Phe NH₂ (SEQ ID No. 336) H- Arg Ser Ile Gly pFF NH₂ (SEQ ID No. 337) H- Arg Ser Ile Gly mClF NH₂ (SEQ ID No. 338) H- Arg Ser Val Asn Phe NH₂ (SEQ ID No. 339) H- Arg Ser Val Asn pFF NH₂ (SEQ ID No. 340) H- Arg Ser Val Asn mClF NH₂ (SEQ ID No. 341) H- Arg Ser Val Ala Phe NH₂ (SEQ ID No. 342) H- Arg Ser Val Ala pFF NH₂ (SEQ ID No. 343) H- Arg Ser Val Ala mClF NH₂ (SEQ ID No. 344) H- Arg Ser Val Gly Phe NH₂ (SEQ ID No. 345) H- Arg Ser Val Gly pFF NH₂ (SEQ ID No. 346) H- Arg Ser Val Gly mClF NH₂ (SEQ ID No. 347) H- Arg Lys Leu Asn Phe NH₂ (SEQ ID No. 348) H- Arg Lys Leu Asn pFF NH₂ (SEQ ID No. 349) H- Arg Lys Leu Asn mClF NH₂ (SEQ ID No. 350) H- Arg Lys Leu Ala Phe NH₂ (SEQ ID No. 351) H- Arg Lys Leu Ala pFF NH₂ (SEQ ID No. 352) H- Arg Lys Leu Ala mClF NH₂ (SEQ ID No. 353) H- Arg Lys Leu Gly Phe NH₂ (SEQ ID No. 354) H- Arg Lys Leu Gly pFF NH₂ (SEQ ID No. 355) H- Arg Lys Leu Gly mClF NH₂ (SEQ ID No. 356) H- Arg Lys Ile Asn Phe NH₂ (SEQ ID No. 357) H- Arg Lys Ile Asn pFF NH₂ (SEQ ID No. 358) H- Arg Lys Ile Asn mClF NH₂ (SEQ ID No. 359) H- Arg Lys Ile Ala Phe NH₂ (SEQ ID No. 360) H- Arg Lys Ile Ala pFF NH₂ (SEQ ID No. 361) H- Arg Lys Ile Ala mClF NH₂ (SEQ ID No. 362) H- Arg Lys Ile Gly Phe NH₂ (SEQ ID No. 363) H- Arg Lys Ile Gly pFF NH₂ (SEQ ID No. 364) H- Arg Lys Ile Gly mClF NH₂ (SEQ ID No. 365) H- Arg Lys Val Asn Phe NH₂ (SEQ ID No. 366) H- Arg Lys Val Asn pFF NH₂ (SEQ ID No. 367) H- Arg Lys Val Asn mClF NH₂ (SEQ ID No. 368) H- Arg Lys Val Ala Phe NH₂ (SEQ ID No. 369) H- Arg Lys Val Ala pFF NH₂ (SEQ ID No. 370) H- Arg Lys Val Ala mCIF NH₂ (SEQ ID No. 371) H- Arg Lys Val Gly Phe NH₂ (SEQ ID No. 372) H- Arg Lys Val Gly pFF NH₂ (SEQ ID No. 373) H- Arg Lys Val Gly mClF NH₂ (SEQ ID No. 374) H- Arg Arg Leu Ile pFF NH₂ (SEQ ID No. 375) H- Cit Cit Leu Ile pFF NH₂ (SEQ ID No. 376) H- Arg Arg Leu Ile Phe NH₂ (SEQ ID No. 377)

[0184] More preferably still, the invention relates to peptides of formula V which are selected from the following: H- Arg Arg Leu Asn Phe NH₂ (SEQ ID No. 294) H- Arg Arg Leu Asn pFF NH₂ (SEQ ID No. 295) H- Arg Arg Leu Asn mClF NH₂ (SEQ ID No. 296) H- Arg Arg Leu Ala pFF NH₂ (SEQ ID No. 298) H- Arg Arg Leu Ala mClF NH₂ (SEQ ID No. 299) H- Arg Arg Leu Gly pFF NH₂ (SEQ ID No. 301) H- Arg Arg Leu Gly mClF NH₂ (SEQ ID No. 302) H- Arg Arg Ile Asn pFF NH₂ (SEQ ID No. 304) H- Arg Arg Ile Asn mClF NH₂ (SEQ ID No. 305) H- Arg Arg Ile Ala pFF NH₂ (SEQ ID No. 307) H- Arg Arg Ile Ala mClF NH₂ (SEQ ID No. 308) H- Arg Lys Leu Asn mClF NH₂ (SEQ ID No. 350) H- Arg Lys Leu Ala pFF NH₂ (SEQ ID No. 352) H- Arg Lys Leu Ala mClF NH₂ (SEQ ID No. 353) H- Arg Lys Leu Gly pFF NH₂ (SEQ ID No. 355) H- Arg Lys Ile Asn pFF NH₂ (SEQ ID No. 358) H- Arg Arg Leu Ile pFF NH₂ (SEQ ID No. 375)

[0185] In one especially preferred embodiment, the peptides of formula V are selected from the following: H- Arg Arg Leu Asn Phe NH₂ (SEQ ID No. 294) H- Arg Arg Leu Asn pFF NH₂ (SEQ ID No. 295) H- Arg Arg Leu Asn mClF NH₂ (SEQ ID No. 296) H- Arg Arg Leu Ala pFF NH₂ (SEQ ID No. 298) H- Arg Arg Ile Asn pFF NH₂ (SEQ ID No. 304) H- Arg Arg Ile Ala pFF NH₂ (SEQ ID No. 307) H- Arg Lys Leu Ala pFF NH₂ (SEQ ID No. 352) H- Arg Arg Leu Asn pFF NH₂ (SEQ ID No. 295) H- Arg Arg Ile Asn pFF NH₂ (SEQ ID No. 304) H- Arg Arg Leu Ile pFF NH₂ (SEQ ID No. 375)

[0186] The present invention further encompasses the above described peptides of the first, second and third aspects, their use in the inhibition of CDK2, their use in the treatment of proliferative disorders such as cancers and leukaemias where inhibition of CDK2 would be beneficial and their use in the preparation of medicaments for such use. Such preparation including their use in assays for further candidate compound as described herein. The embodiments described as being preferred in the context of the peptides of the invention apply equally to their use.

[0187] Assays

[0188] A further embodiment of the present invention relates to assays for candidate substances that are capable of modifying the cyclin interaction with CDK's, especially CDK2 and CDK4. Such assays are based upon the observation that the peptides of the invention, despite not including the generally considered “cyclin binding motif” as discussed in Example 9, have been shown to bind to cyclin. Furthermore, it has been shown that the peptides of the second and further aspects of the invention competitively inhibit the binding of a peptide of the first aspect of the invention. Thus, such assays may involve incubating a candidate substance with a cyclin and a peptide of the invention and detecting either the candidate-cyclin complex or free (unbound) peptide of the invention., An example of the latter would involve the peptide of the invention being labeled such as to emit a signal when bound to a CDK. The reduction in said signal being indicative of the candidate substance binding to, or inhibiting peptide-cyclin interaction.

[0189] Suitable candidate substances include peptides, especially of from about 5 to 30 or 10 to amino acids in size, based on the sequence of the various domains of p21, or variants of such peptides in which one or more residues have been substituted. Peptides from panels of peptides comprising random sequences or sequences which have been varied consistently to provide a maximally diverse panel of peptides may be used.

[0190] Suitable candidate substances also include antibody products (for example, monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies and CDR-grafted antibodies) which are specific for p21 or cyclin binding regions thereof. Furthermore, combinatorial libraries, single-compound collections of synthetic or natural organic molecules, peptide and peptide mimetics, defined chemical entities, oligonucleotides, and natural product libraries may be screened for activity as modulators of cyclin/CDK/regulatory protein complex interactions in assays such as those described below. The candidate substances may be used in an initial screen in batches of, for example, 10 substances per reaction, and the substances of those batches which show inhibition tested individually. Candidate substances which show activity in in vitro screens such as those described below can then be tested in whole cell systems, such as mammalian cells.

[0191] Thus the present invention further relates to an assay for the identification of compounds that interact with cyclin A, cyclin E or cyclin D (hereinafter “a cyclin”) or these cyclins when complexed with the physiologically relevant CDK, comprising;

[0192] (a) incubating a candidate compound and a peptide of the formula X₁X₂X₃RX₄LX₅F (SEQ ID No. 2) or more preferably of formula HX₂KRRLX₅F (SEQ ID No.3) or variants thereof as defined above, and a cyclin or cyclin/CDK complex,

[0193] (b) detecting binding of either the candidate compound or the peptide of formula X₁X₂X₃RX₄LX₅F (SEQ ID No. 2)/HX₂KRRLX₅F (SEQ ID No. 3) with the cyclin.

[0194] The assays of the present invention (discussed hereinafter with reference to cyclin A) encompass screening for candidate compounds that bind a cyclin “recruitment center” or “cyclin groove” discussed above in respect of the prior art but herein defined in greater detail with reference to the amino acid sequence of preferably human cyclin A or of partially homologous and functionally equivalent mammmalian cyclins. The substrate recruitment site from previously described cyclin A/peptide complexes consists mainly of residues of the α1 (particularly residues 207-225) and α3 (particularly residues 250-269) helices, which form a shallow groove on the surface, comprised predominantly of hydrophobic residues. This is discussed in greater detail in Russo A A et al. (Nature (1996) 382, 325-331) with respect to p27/cyclin A. From the X-ray structure assigned to the p27/cyclin A/CDK2 provided therein it is possible to conclude that the sequence SACRNLFG (SEQ ID No. 178) of p27 that interacts with cyclin A does so through the following interaction residues of cyclin A: p27 residue Cyclin A residues S E220, E224 A W217, E220, V221, E224, I281 C Y280, I281, D283 R D216, W217, E220, Q254 N Q254, T285, Y286 L I213, L214, W217, Q254 F M210, I213, R250, G251, K252, L253, Q254 G T285

[0195] These residues are largely conserved in the A, B, E and D1 cyclins.

[0196] Through analysis of the interaction of the p21 peptides of the present invention with cyclin A, further distinct amino acid residues of cyclin A have been identified as being important in the interaction between cyclin A and p21, especially with respect to the inhibitory activity the peptides of the present invention display against CDK2.

[0197] The cyclin A amino acids believed to be important for interaction with the p21 derived peptides of the present invention include: Cyclin A residues Major Intermediate Minor p21 residue Interaction Interaction Interaction H E223, E224 W217, V219, V221 G222, Y225, I281 S408, E411 A Y225 E223 K D284 E220, V279 R I213 A212, V215, L218 Q406, S408 R D283 I213, L214 M210, L253 L L253 G257 L218, I239, V256 I R250, Q254 F I206, R211 T207, L214 M200

[0198] The present invention therefore includes assays for candidate compounds that interact with cyclin A by virtue of forming associations with at least two of the amino acid residues L253, 1206 and R211 of cyclin A or the corresponding homologous amino acids of cyclin D or cyclin E.

[0199] In a further preferred assay, the candidate compound may form associations with at least E223, E224, D284, D283, L253, 1206 and R211 of cyclin A or the corresponding homologous amino acids of cyclin D or cyclin E.

[0200] In a preferred assay, the candidate compound may form further associations with W217, V219, V221, S408, E411, Y225, 1213, L214, G257, R250, Q254, T207 and L214 of cyclin A or the corresponding homologous amino acids of cyclin D or cyclin E.

[0201] In a more preferred assay, the candidate compound may form further associations with G222, Y225, 1281, E223, E220, V279, A212, V215, L218, Q406, S408, M210, L253, L218, 1239, V256 and M200 of cyclin A or the corresponding homologous amino acids of cyclin D or cyclin E.

[0202] As used in this context the phrase “forming associations” is used to include any form of interaction a binding peptide may make with a peptide ligand. These include electrostatic interactions, hydrogen bonds, or hydrophobic/lipophilic interactions through Van der Waals's forces or aromatic stacking, etc.

[0203] Also, as used herein in the context of assays of the present invention, the term “cyclin” is used to refer to cyclin A, cyclin D or cyclin E, or regioins thereof that incorporate the “cyclin groove” as hereinbefore described. Thus, an assay may be performed in accordance with the present invention if it utilises the a full length cyclin protein or a region sufficient to allow the cyclin groove to exist, for example amino acids 173-432 or 199-306 of human cyclin A.

[0204] Thus, by utilising the peptides of the present invention especially those of the preferred embodiments in competitive binding assays with candidate compounds, further compounds that interact at this site may be identified and assigned utility in the control of the cell cycle by virtue of controlling, preferably inhibiting CDK2 and/or CDK4 activity. Such assays may be performed in vitro or virtually i.e. by using a three dimensional model or preferably, a computer generated model of a complex of a peptide of the present invention and cyclin A. Using such a model, candidate compounds may be designed based upon the specific interactions between the peptides of the present invention and cyclin A, the relevant bond angles and orientation between those components of the peptides of the present invention that interact both directly and indirectly with the cyclin groove. By way of example, FIG. 4 shows the interaction between the peptide HAKRRLIF (SEQ ID No. 42) and Cyclin A. From using the three dimensional model computer generated by this interaction it has been possible to identify the cyclin A amino acid residues that interact with the peptides of the present invention, particularly with HAKRRLIF (SEQ ID No. 42) as outlined above and discussed in greater detail in the examples.

[0205] In an embodiment, the cyclin groove includes about residues 173-432 of human cyclin A. In another embodiment, the cyclin groove includes about residues 199-306 of human cyclin A. In a preferred embodiment, the cyclin groove includes about residues 207-225 and about residues 250-269 and about residues 274-282 of human cyclin A. In another embodiment, the cyclin groove includes one or more of: about residues 207-225; about residues 250-269; and about residues 274-282 of human cyclin A. In another embodiment, the cyclin groove includes two or more of: about residues 207-225; about residues 250-269; and about residues 274-282 of human cyclin A

[0206] As used herein the term “three dimensional model” includes both crystal structures as determined by X-ray diffraction analysis, solution structures determined by nuclear magnetic resonance spectroscopy as well as computer generated models. Such computer generated models may be created on the basis of a physically determined structure of a peptide of the present invention bound to cyclin A or on the basis of the known crystal structure of cyclin A, modified (by the constraints provided by the software) to accommodate a peptide of formula I. Suitable software suitable of the generation of such computer generated three dimensional models include AFFINITY, CATALYST and LUDI (Molecular Simulations, Inc.).

[0207] Such three dimensional models may be used in a program of rational drug design to generate further candidate compounds that will bind to cyclin A. As used herein the term “rational drug design” is used to signify the process wherein structural information about a ligand-receptor interaction is used to design and propose modified ligand candidate compounds possessing improved fit with the receptor site in terms of geometry and chemical complementarity and hence improved biological and pharmaceutical properties, such properties including, e.g., increased receptor affinity (potency) and simplified chemical structure. Such candidate compounds may be further compounds or synthetic organic molecules. The preferred peptides for use in these aspects of the invention are identical to those designated as preferred with respect to the first and second aspects of the invention, most especially those of the formula HX₂KRRLX₅F (SEQ ID No.3) and of those particularly the peptide HAKRRLIF (SEQ ID No. 42). In a preferred embodiment, rational drug design is focussed upon the four C-terminal amino acids RLX₅F (SEQ ID No. 184) or RLFX₅ (SEQ ID No. 185) or variants thereof as discussed above with respect to SEQ ID No. 3.

[0208] Using techniques known in the art, crystal or solution structures of cyclin A bound to a peptide of the present invention may be generated, these too may be used in a programme of rational drug design as discussed above.

[0209] Crystals of the p21 derived peptides of the present invention complexed with cyclin A can be grown by a number of techniques including batch crystallization, vapor diffusion (either by sitting drop or hanging drop) and by microdialysis. Seeding of the crystals in some instances is required to obtain X-ray quality crystals. Standard micro and/or macro seeding of crystals may therefore be used.

[0210] Once a crystal of the present invention is grown, X-ray diffraction data can be collected. Crystals can be characterized by using X-rays produced in a conventional source (such as a sealed tube or a rotating anode) or using a synchrotron source. Methods of characterization include, but are not limited to, precision photography, oscillation photography, diffractometer data collection, and Se-Met multiwavelength anamalous dispersion data.

[0211] Once the three-dimensional structure of a protein-ligand complex formed between a p21 derived peptide of the present invention and cyclin A is determined, a candidate compound may be examined through the use of computer modeling using a docking program such as GRAM, DOCK or AUTODOCK [Dunbrack et al., 1997, Folding & Design 2:R27-42]. This procedure can include computer fitting of candidate compounds to the ligand binding site to ascertain how well the shape and the chemical structure of the candidate compound will complement the binding site. [Bugg et al., Scientific American, December:92-98 (1993); West et al;l TIPS, 16:67-74 (1995)]. Computer programs can also be employed to estimate the attraction, repulsion and steric hindrance of the two binding partners (i.e. the ligand-binding site and the candidate compound). Generally the tighter the fit, the lower the steric hindrances, and the greater the attractive forces, the more potent the potential drug since these properties are consistent with a tighter binding constant. Furthermore, the more specificity in the design of a potential drug the more likely that the drug will not interact as well with other proteins. This will minimize potential side-effects due to unwanted interactions with other proteins.

[0212] Initially candidate compounds can be selected for their structural similarity to a p21 derived peptide of the present invention such as HAKRRLIF (SEQ ID No. 42), the four C-terminal amino acids thereof RLX₅F (SEQ ID No. 184) or RLFX₅ (SEQ ID No. 185); or variants or a region thereof. The structural analog can then be systematically modified by computer modeling programs or by inspection until one or more promising candidate compounds are identified. A candidate compound could be obtained by initially screening a random peptide library produced by recombinant bacteriophage for example [Scott and Smith, Science, 249:386-390 (1990); Cwirla et al., Proc. Natl. Acad. Sci., 87:6378-6382 (1990); Devlin et al., Science, 249:404-406 (1990)]. A peptide selected in this manner would then be systematically modified by computer modeling programs as described above, and then treated analogously to a structural analog as described below.

[0213] Once a candidate compound is identified it can be either selected from a library of chemicals as are commercially available or, alternatively, the candidate compound or antagonist may be synthesized de novo. As mentioned above, the de novo synthesis of one or even a relatively small group of specific compounds is reasonable in the art of drug design. The candidate compound can be placed into a standard binding assay with cyclin A together with a peptide of the present invention and its relative activity assessed.

[0214] In such an assay, cyclin A may be attached to a solid support. Methods for placing such a binding domain on the solid support are well known in the art and include such things as linking biotin to the ligand binding domain and linking avidin to the solid support. The solid support can be washed to remove unreacted species. A solution of a labeled candidate compound alone or together with a peptide of the present invention can be contacted with the solid support. The solid support is washed again to remove the candidate compound/peptide not bound to the support. The amount of labeled candidate compound remaining with the solid support and thereby bound to the ligand binding domain may be determined. Alternatively, or in addition, the dissociation constant between the labeled candidate compound and cyclin A can be determined. Alternatively, if a peptide of the present invention is used, it may be labeled and the decrease in bound labeled peptide used an indication of the relative activity of the candidate compound. Suitable labels are exemplified in our WO00/50896 (the contents of which are hereby incorporated by reference) which describes suitable fluorescent labels for use in fluorescent polarisation assays for protein/protein and protein/non-protein binding reactions. Such assay techniques are of use in the assays and methods of the present invention.

[0215] When suitable candidate compounds are identified, a supplemental crystal may be grown comprising a protein-candidate complex formed between cyclin A and the potential drug. Preferably the crystal effectively diffracts X-rays for the determination of the atomic coordinates of the protein-candidate complex to a resolution of greater than 5.0 Angstroms, more preferably greater than 3.0 Angstroms, and even more preferably greater than 2.0 Angstroms. The three-dimensional structure of the supplemental crystal may be determined by Molecular Replacement Analysis. Molecular replacement involves using a known three-dimensional structure as a search model to determine the structure of a closely related molecule or protein-candidate complex in a new crystal form. The measured X-ray diffraction properties of the new crystal are compared with the search model structure to compute the position and orientation of the protein in the new crystal.

[0216] Computer programs that can be used include: X-PLOR (Bruger X-PLOR v.3.1Manual, New Haven: Yale University (1993B)) and AMORE [J. Navaza, Acta Crystallographics ASO, 157-163 (1994)]. Once the position and orientation are known an electron density map can be calculated using the search model to provide X-ray phases. Thereafter, the electron density is inspected for structural differences and the search model is modified to conform to the new structure.

[0217] Candidates whose cyclin A binding capability has thus been verified biochemically can then form the basis for additional rounds of drug design through structure determination, model refinement, synthesis, and biochemical screening all as discussed above, until lead compounds of the desired potency and selectivity are identified. The candidate drug is then contacted with a cell that expresses cyclin A. A candidate drug is identified as a drug when it inhibits CDK2 and/or CDK4 in the cell. The cell can either by isolated from an animal, including a transformed cultured cell; or alternatively can be present in a living animal.

[0218] In such assays, and as alternative embodiments of the herein described assays, a functional end-point may be monitored as an indication of efficacy in preference to the detection of cyclin binding. Such end-points include: G0 and/or G1/S cell cycle arrest (using flow cytometry), cell cycle-related apoptosis (sub-G0 population by fluorescence-activated cell sorting, FACS; or TUNEL assay), suppression of E2F transcription factor activity (e.g. using a cellular E2F reporter gene assay), hypophosphorylation of cellular pRb (using Western blot analysis of cell lysates with relevant phospho-specific antibodies), or generally in vitro anti-proliferative effects.

[0219] Thus, a further related aspect of the present invention relates to a three dimensional model of a peptide of the formula X₁X₂X₃RX₄LX₅F (SEQ ID No. 2) or preferably HX₂KRRLX₅F (SEQ ID No. 3): or variants thereof as defined above and cyclin A.

[0220] The invention further includes a method of using a three-dimensional model of cyclin A and a peptide of the present invention in a drug screening assay comprising;

[0221] (a) selecting a candidate compound by performing rational drug design with the three-dimensional model, wherein said selecting is performed in conjunction with computer modeling;

[0222] (b) contacting said candidate compound with cyclin A, and

[0223] (c) detecting the binding of the candidate compound; wherein a potential drug is selected on the basis of the candidate compound having a similar or greater affinity for cyclin A than that of a standard p21 derived peptide.

[0224] In a preferred embodiment the standard p21 derived peptide has the general formula HX₂KRRLX₅F (SEQ ID No. 3): as defined above. Preferably, the three dimensional model is a computer generated model.

[0225] The peptides of the invention and substances identified or identifiable by the assay methods of the invention may preferably be combined with various components to produce compositions of the invention. Preferably the compositions are combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition (which may be for human or animal use). Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline. The composition of the invention may be administered by direct injection. The composition may be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration. Typically, each protein may be administered at a dose of from 0.01 to 30 mg/kg body weight, preferably from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight.

[0226] Pharmaceutically acceptable salts of the peptides of the invention include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric and maleic. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine and procaine.

[0227] Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% to 95% of active ingredient, preferably 25% to 70%. Where the vaccine composition is lyophilised, the lyophilised material may be reconstituted prior to administration, e.g. as a suspension. Reconstitution is preferably effected in buffer

[0228] Capsules, tablets and pills for oral administration to a patient may be provided with an enteric coating comprising, for example, Eudragit “S”, Eudragit “L”, cellulose acetate, cellulose acetate phthalate or hydroxypropylmethyl cellulose.

EXAMPLES Abbreviations

[0229] The nomenclature for amino acid and peptide derivatives conforms with IUPAC-IUB rules (J. Peptide Sci. 1999, 5, 465-471). D-amino acids are indicated by lower-case abbreviations, e.g. Ala for L-alanine, ala for D-alanine. Non-standard abbreviations for amino-acid residues are as follows: Abu 2-Aminobutyric acid

Aib Aminoisobutyric acid

Ahx ε-Aminohexanoic acid

hArg Homoarginine

Bug t-Butylglycine

oClPhe o-Chlorophenylalanine

mClPhe m-Chlorophenylalanine

pClPhe p-Chlorophenylalanine

Cha Cyclohexylalanine

DiClPhe m,p-Dichlorophenylalanine

Cit Citrulline

Dhp Dehydrophenylalanine

Dab 1,3-Diaminobutyric acid

mFPhe m-Fluorophenylalanine

pFPhe p-Fluorophenylalanine

Hof Homophenylalanine

Hse alle Homoserine allo-Isoleucine

Inc 2-Indolecarboxylic acid

pIPhe p-Iodophenylalanine

1Nap 1-Naphthylalanine

2Nap 2-Naphthylalanine

Nle Norleucine

Nva Norvaline

Pheol Phenylalaninol

Phg Phenylglycine

Psa O-Acetylphenylserine

Pse Phenylserine

Pya 3-Pyridylalanine

Sar Sarcosine

Thi 2-Thienylalanine

Tic 1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid

Tyr(Me) O-Methyltyrosine

[0230] Other abbreviations used: Boc t-Butyloxycarbonyl BSA Bovine serum albumin CDK Cyclin-dependent kinase DE MALDI- Delayed extraction matrix-assisted TOF MS laser desorption ionisation time-of-flight mass spectrometry DMF Dimethylformamide ES-MS Electrospray ionisation mass spectrometry FAB-MS Fast atom bombardment mass spectrometry Fmoc Fluoren-9-ylmethoxycarbonyl Fmoc-ONSu Fmoc N-hydroxysuccinimidyl ester HBTU 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate HOBt 1-Hydroxybenzotriazole IC₅₀ Concentration at which 50% inhibition is observed Mtt 4-Methyltrityl NMP N-methylpyrrolidinone Pbf 2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl Pmc 2,2,5,7,8-Pentamethylchroman-6-sulfonyl PyBOP Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate RP-HPLC Reversed-phase high-performance liquid chromatography TBTU 2-(1H-Benzatriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate TFA Trifluoroacetic acid THF Tetrahydrofuran TLC Thin layer chromatography Trt Trityl

[0231] Example 1

Peptide Inhibitors of Rb Phosphorylation by G1 CDKs

[0232] Experimental Procedures

[0233] Unless otherwise indicated, the peptides in the examples below were assembled using a Multipin Peptide Synthesis Kit (Chiron Technologies, Clayton, VIC, Australia; Valerio, R. M.; Bray, A. M.; Maeji, N. J. Intl. J. Peptide Protein Res. 1994, 44, 158-165 & Valerio et al., 1993) or an automated peptide synthesiser (ABI 433A). In either case, the solid-phase linker was 4-(2′,4-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamido (Rink amide linker; Rink, H. Tetrahedron Lett. 1987, 28, 3787-3790 & Fields et al. 1990). Standard solid-phase chemistry based on the Fmoc protecting group (Atherton, E.; Sheppard, R. C.

[0234] Solid phase peptide synthesis: a practical approach; IRL Press at Oxford University Press: Oxford, 1989) was employed using PyBOP-HBTU- or TBTU-mediated acylation chemistry in the presence of HOBt and Pr₂ ^(i)NEt, in either NMP or DMF. Repetitive Fmoc-deprotection was achieved with piperidine. The following amino acid side-chain protecting groups were used: Asp(OBu^(t)), Glu(OBu^(t)), His(Trt), Lys(Boc), Arg(Pmc), Hse(Bu^(t)), Ser(Bu^(t)), Dab(Boc), Asn (Trt), Gln(Trt), Trp(Boc). Peptides were side-chain deprotected and cleaved from the synthesis support using either of the following acidolysis mixtures: a) 2.5:2.5:95 (v/v/v) Pr₃ ^(i)SiH, H₂O, CF₃COOH, b) 0.75:0.5:0.5:0.25:10 (w/v/v/v/v) PhOH, PhSMe, H₂O, HSCH₂CH₂SH, CF₃COOH (King et al., 1990). Cleavage/deprotection was allowed to proceed for 2.5 h under N₂, before evaporation in vacuo, precipitation from Et₂O, and drying. All peptides were purified by preparative RP-HPLC or solid phase extraction (on octadecylsilane cartridges), isolated by lyophilisation, and were analyzed by analytical RP-HPLC and mass spectrometry (Dynamo DE MALDI-TOF spectrometer, ThermoBioAnalysis).

[0235] Peptide synthesis. Peptides were assembled using a Multipin Peptide Synthesis Kit (Chiron Technologies, Clayton, VIC, Australia) (Valerio et al., 1993). Standard solid-phase chemistry based on the Fmoc protecting group was employed (Fields et al., 1990). Peptides were side-chain deprotected and cleaved from the synthesis support using methods as described (King et al., 1990). All peptides were purified by preparative reversed-phase HPLC or solid phase extraction, isolated by lyophilisation, and were analyzed by analytical HPLC and mass spectrometry (Dynamo DE MALDI-TOF spectrometer, ThermoBioAnalysis).

Example 2 Production of Recombinant Proteins

[0236] PKCα-6×His, CDK4-6×His, CDK2-6×His/Cyclin E-6×His, CDK1-6×His/Cyclin B-6×His-His-tagged CDK2/Cyclin E and CDK1/Cyclin B were co-expressed and PKCα, and CDK4 were singularly expressed in Sf 9 insect cells infected with the appropriate baculovirus constructs. The cells were harvested two days after infection by low speed centrifugation and the proteins were purified from the insect cell pellets by Metal-chelate chromatography. Briefly, the insect cell pellet was lysed in Buffer A (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.02% NP40 and 5 mM β-marcaptoethanol, 1 mM NaF. 1 mM Na3VO4 and Protease Inhibitors Coctail (Sigma) containing AEBSF, pepstatin A, E 64, bestatin, leupeptin) by sonication. The soluble fraction was cleared by centrifugation and loaded onto Ni-NTA-Agarose (Quiagen). Non bound proteins were washed off with 300 mM NaCl, 5-15 mM Imidazole in Buffer A and the bound proteins were eluted with 250 mM Imidazole in Buffer A. The purified proteins were extensively 1.5 dialyzed against Storage buffer (20 mM HEPES pH 7.4, 50 mM NaCl, 2 mM DTT, 1 mM EDTA, 1 mM EGTA, 0.02% NP40, 10% v/v Glycerol) aliquoted and stored at −70° C. PKC-α-6×His was purified the same way but using different buffers-50 mM NaH2PO4, pH 8.0 and 0.05% Triton X-100 instead of Tris and NP40 respectively.

[0237] Cyclin D1 and p21 were expressed in E coli BL21 (DE3) using PET expression vectors. BL21 (DE3) was grown at 37° C. with shaking (200 rpm) to mid-log phase (OD600 nm=0.6). Expression was induced by the addition of IPTG at a final concentration of 1 mM, and the culture was incubated for a further 3 h. The bacteria were than harvested by centrifugation, and the cell pellet was resuspended in 50 mM Tris-HCl, pH 7.5, 10% sucrose. Both proteins were purified from inclusion bodes. Briefly, the bacterial cells were lysed by treatment with lysosyme and sonication. The insoluble fraction was pelleted by centrifugation. The inclusion bodies were purified by repetitive washing of the insoluble fraction with 50 mM Tris-Hcl pH 8.0, 2 mM EDTA, 100 mM NaCl and 0.5% Triton X-100. Purified inclusion bodies were solubilized with the same buffer, containing 6M urea. The proteins were refolded by slow dilution with 25 mM Tris-HCl pH 8.0, 100 mM NaCl, 2 mM DTT, 1 mM EDTA, 0.2% NP40. After concentration by ultrafiltration (Amicon concentration unit) the purified proteins were aliquoted and stored at −70° C.

[0238] GST-Rb—An E coli expression construct containing the hyperphosphorylation domain of pRb (amino acids 773-924) was purified on a Glutathione-Sepharose column according to the manufacturers instructions (Pharmacia). For the 96-well format “in vitro” kinase assay GST-Rb was used immobilized on Glutathione-Sepharose beads.

Example 3 Enzyme Assays

[0239] CDK4/Cyclin D1, CDK2/Cyclin E, CDIK1/Cyclin B Kinase Assays

[0240] Phosphorylation of GST-Rb

[0241] GST-Rb phosphorylation, induced by CDK4/Cyclin D1, CDK2/Cyclin E or CDK1/Cyclin B was determined by incorporation of radio-labeled phosphate in GST-Rb(772-928) using radiolabelled ATP in 96-well format in vitro kinase assay. The phosphorylation reaction mixture (total volume 40 μl) consisted of 50 mM HEPES pH 7.4, 20 mM MgCl2, 5 mM EGTA, 2 mM DTT, 20 mM β-glycerophosphate, 2 mM NaF, 1 nM Na3VO4, Protease Inhibitors Cocktail (Sigma, see above), BSA 0.5 mg/ml, 1 μg purified enzyme complex, 10 μl of GST-Rb-Sepharose beads, 100 μM ATP, 0.2 μCi ³²P-ATP. The reaction was carried out for 30 min at 30° C. at constant shaking. At the end of this period 100 μl of 50 mM HEPES, pH 7.4 and 1 mM ATP were added to each well and the total volume was transferred onto GFC filtered plate. The plate was washed 5 times with 200 μl of 50 mM HEPES, pH 7.4 and 1 mM ATP. To each well were added 50 μl scintillant liquid and the radioactivity of the samples was measured on Scintilation counter (Topcount, HP). The IC50 values of different peptides were calculated using GraFit software.

[0242] Phosphorylation of Histone

[0243] Histone 1 phosphorylation induced by CDK2/Cyclin E and CDK1/Cyclin B was measured using similar method. The concentration of Histone 1 in the kinase reaction was 1 mg/ml (unless different stated). The kinase reaction was stopped by 75 mM Phosphoric acid (100 μl per well) and the reaction mixture was transferred onto P81 plates. The plates were washed 3 times with 200 μl 75 mM orthophosphoric acid.

[0244] Protein Kinase C (PKC)α Assay

[0245] PKCαkinase activity was measured by the incorporation of radio-labeled phosphate in Histone 3. The reaction mixture (total volume 65 μl) consist of 50 mM Tris-HCl, 1 mM Calcium acetate, 3 mM DTT, 0.03 mg/ml Phosphatidylserine, 2.4 μg/ml PMA, 0.04% NP40, 12 mM Mg/Cl, purified PKCα-100 ng, Histone 3, 0.2 mg/ml, 100 μM ATP, 0.2 μCi [γ-³²P]-ATP. The reaction was carried over 15 min at 37° C. in microplate shaker and was stopped by adding 10 μl 75 mM orthophosphoric acid and placing the plate on ice. 50 μl of the reaction mixture was transferred onto P81 filterplate and after washing off the free radioactive phosphate (3 times with 200 μl 75 mM orthophosphoric acid per well) 50 μl of scintillation liquid (Microscint 40) were added to each well and the radioactivity was measured on Scintillation counter (1 opcount, HP).

[0246] ERK-2 (MAP Kinase) Assay

[0247] ERK-2 kinase activity was measured by the incorporation of radio-labeled phosphate into Myelin Basic Protein (MBP), catalyzed by purified mouse ERK2 (Upstate Biotecnoligies). The reaction mixture (total volume 50 μl) consisted of 20 mM MOPS, pH 7.0, 25 mM β-glycerophosphate, 5 mM EGTA, 1 mM DTT, 1 mM Na₃VO₄, 10 mM MgCl, 100 μM ATP, 0.2 μCi [γ-³²P]-ATP.

[0248] CDK2/Cyclin A

[0249] CDK2/cyclin A kinase assays were performed in 96-well plates using recombinant CDK2/cyclin A. Assay buffer consisted of 25 mM β-glycerophosphate, 20 mM MOPS, 5 mM EGTA, 1 mM DTT, 1 mM NaVO₃, pH 7.4, into which was added 2-4 μg of CDK2/cyclin A with substrate pRb(773-928). The reaction was initiated by addition of Mg/ATP mix (15 mM MgCl₂, 100 μM ATP with 30-50 kBq per well of [γ-³²P]-ATP) and mixtures incubated for 10-30 min, as required, at 30° C. Reactions were stopped on ice, followed by filtration through p81 filterplates (Whatman Polyfiltronics, Kent, UK). After washing 3 times with 75 mM orthophosphoric acid, plates were dried, scintillant added and incorporated radioactivity measured in a scintillation counter (TopCount, Packard Instruments, Pangbourne, Berks, UK).

[0250] Competitive Cyclin D1/Cyclin A Binding Assay (ELISA).

[0251] Biotinylated p21 (149-159)-DFYHSKRRLIF (SEQ ID No. 1) was immobilized on Streptavidin coated 96-well plates (PIERCE). Different amounts of a competitor peptide were mixed with Cyclin D1/Cyclin A and than loaded onto the plate with immobilized biotinylated p21 (149-159). The amount of bound Cyclin D1/Cyclin A was immunodetected and quantified by Turbo-ELISA reagent (PIERCE). The IC 50 values (a concentration of the competitor peptide which inhibits 50% of Biotin-p21 (149-159)-Cyclin D1/Cyclin A binding) were calculated using GraFit software.

[0252] Cyclin A Binding Assay

[0253] Streptavidin-coated plates (Reacti-Bind™, Pierce) were washed three times with TBS/BSA buffer (25 mM Tris·HCl, 150 mM NaCl pH 7.5, 0.05% Tween-20, 0.1% BSA; 200 μL) for 2 min each. A 10 mM stock solution of biotinyl-Ahx-His-Ala-Lys-Arg-Arg-Leu-Ile-Phe-NH₂ (SEQ ID No. 186) was diluted to 0.5 μM with TBS/BSA buffer. This was added to each well (100 μL). The plate was incubated for 1 h at room temperature with constant shaking. The plate was washed once quickly with TBS/BSA buffer (200 μL), followed by three more washes with TBS/BSA buffer (200 μL) for 5 min each. Serial dilutions of test peptides were prepared in a new plate (50 μL in each well). Cyclin A was diluted to 5 μg/50 μL with TBS/BSA buffer and this was then added to each well (50 μL). The solutions were mixed thoroughly with a pipette (5-6 times), before being incubated for 30 min at room temperature. This reaction mixture was then transferred to the biotinylated peptide:streptavidin-coated plate and incubated for 1 h at room temperature with constant shaking. The plate was washed once quickly with TBS/BSA buffer (200 μL), followed by three more washes with TBS/BSA buffer (200 μL) for 5 min each. The cyclin A antibody (Santa Cruz polyclonal) solution was diluted 1:200 with TBS/BSA buffer and this was then added to each well of the plate (100 μL. The plate was incubated for 1 h at room temperature with constant shaking. The plate was washed once quickly with TBS/BSA buffer (200 μl), followed by three more washes with TBS/BSA buffer (200 μL) for 5 min each. The anti-rabbit secondary antibody (goat anti-rabbit IgG peroxidase conjugate) was diluted 1:10,000 with TBS/BSA and this was then added to each well of the plate (100 μL). The plate was incubated for 1 h at room temperature with constant shaking. The plate was washed once quickly with TBS/BSA buffer (200 μL), followed by three more washes with TBS/BSA buffer (200 μL) for 5 min each. To each well was added the TMB-ELISA reagent (Pierce 1-Step™ Turbo TMB-ELISA; 100 μL) and the plate incubated for 1 min with constant shaking. The reaction was then quenched by the addition of 2 M aqueous H₂SO₄ (100 μL, each well). The UV absorbance of the each solution was measured spectrophotometrically at 450 nm. IC₅₀ values were calculated from dose-response curves.

Example 4 Molecular Modelling

[0254] The structure co-ordinates of the ternary complex of CDK2/cyclin A/p27^(KIP1) were obtained from the RCSB (accession code 1JSU) and used as the starting point for generating a bound complex of H-His-Ala-Lys-Arg-Arg-Leu-Ile-Phe-NH₂ (SEQ ID No. 36). The peptide was modelled by replacing the residues of the corresponding p27 peptide and manipulating the torsion angles of the Leu-Ile-Phe hydrophobic motif to approximate the bound positioning of the Leu and Phe residues. This structure was then docked into the cyclin groove using the Affinity program (Molecular Simulations, San Diego, Calif.). This molecular docking routine, which incorporates a full molecular mechanics approach, allows for flexibility both in the ligand and in the side chains and backbone of the receptor. For these calculations the side chains and non-α carbons of the cyclin groove were allowed to sample a range of conformational space during optimisation of the peptide/protein complex. The calculation was performed using the CVFF force field, in a two-step process using an implicitly derived solvation model and geometric hydrogen bond restraints. For the initial phase of the calculation, the peptide was minimised into the groove using a simple non-bonded method where the Coulombic and Van der Waals terms are scaled to zero and 0.1, respectively. The subsequent refinement phase involved conformational sampling using molecular dynamics calculated over 5 ps in 100 fs stages, where the temperature is scaled from 500 K to 300 K. The calculation was completed by a final minimisation over 1,000 steps using the Polak-Ribiere Conjugate Gradient method.

Example 5 Structure-Activity Relationships of p21(145-164) Peptides with Respect to Inhibition of Cyclin E/CDK2 and Cyclin D1/CDK4

[0255] Previous studies have shown that a 20-residue peptide, derived from the C-terminus of p21^(WAF1) (residues 141-160) binds to CDK4 and cyclin D1 and is able to inhibit in vitro kinase activity of the CDK4/cyclin D1 complex (Ball, K. L.; Lain, S.; F{dot over (a)}fhraeus, R.; Smythe, C.; Lane, D. P. Curr Biol. 1996, 7, 71-80). In order to define the pharmacophore region of the p21^(WAF1) C-terminus, 12mer overlapping peptides covering the region of p21(145-164) were synthesized. The in vitro effect of these peptides on CDK4/cyclin D1 and CDK2/cyclin E kinase activity in terms of inhibition of phosphorylation of GST-pRb was investigated.

[0256] A shorter sequence being a 12 amino acid peptide DFYHSKRRLIFS—p21 (149-160) (SEQ ID No. 13) was found to have very similar activity as the original 20-mer peptide of Ball et al. with respect to in vitro inhibitory activity in vitro CDK4-Cyclin D1 kinase.

[0257] A detailed SAR analysis of p21 (149-160) was done in 96-well format CDK4-Cyclin D1 kinase assay using different peptide derivatives—truncations and alanine substitutions. In order to determine the relative importance of each position of the 12 amino acid peptide which contained the binding domain, p21(149-160) derivatives were synthesized in which each residue was sequentially substituted with Ala. The effect of the peptide mutations on their kinase inhibitory activity was then tested. Ala substitution of Phe¹⁵⁰, Tyr¹⁵¹, His¹⁵², Ile¹⁵⁸, and Ser¹⁶⁰ did not change significantly the CDK2/cyclin E inhibitory activity of p21(149-160). Substitution of Ser153 with Ala increased 100-fold the inhibitory potency of p21(149-160) towards CDK2/cyclin E. The results are shown in Table 1.

[0258] SAR of P21 (149-160) in CDK2/Cyclin E Kinase Assay.

[0259] P21 (141-160) peptide was shown to inhibit CDK2-Cyclin E induced phosphorylation of GST-Rb (Ball et al., 1995) at concentration 40 times its IC50 of CDK4/cyclin D1. The results herein show that a truncated form—p21 (149-160) and variants thereof, retain very good potency to inhibit the CDK2-Cyclin E induced phosphorylation of GST-Rb and in many cases the peptides were shown to be preferentially inhibitory of CDK2 as opposed to CDK4. Detailed SAR of p21 (149-160) were determined in CDK2-Cyclin E in vitro kinase assay. The data are shown in Table 1.

[0260] A comparison between the SAR of p21 (149-160) in CDK2-Cyclin E and CDK4-Cyclin D1 kinase assays shows a higher inhibitory activity towards CDK2-Cyclin E than to CDK4-Cyclin D1. Alanine mutation of Ser153 increases 100 fold the potency of the peptide to inhibit the CDK2-Cyclin E but has little effect on CDK4-Cyclin D1 induced phosphorylation of GST-Rb. For both inhibitory activities of p21 (149-160) the most important residues are Arg155, Leu 157 and Phe 159. The CDK4-Cyclin D1 inhibitory activity of p21 (149-160) tolerates less changes than the CDK2-Cyclin E one.

[0261] Using identical assays, the sequence p21(148-159) was shown to be active against both CDK2/cyclin E and CDK4/cyclin D1. TABLE 1 Structure-activity relationships of p21 (145-164) peptides with respect to Inhibition of cyclin E/CDK2 and cyclin D1/CDK4 Seq ID P21^(waf1) Sequence^(a) No. Formula M_(r) T S M T D F Y H S K R R L I F S K R K P 187 T S M T D F Y H S K R R 188 C₆₅H₁₀₂N₂₂O₁₉S 1527 71 S M T D F Y H S K R R L 189 C₆₇H₁₀₆N₂₂O₁₈S 1539 76 M T D F Y H S K R R L I 190 C₇₀H₁₁₂N₂₂O₁₇S 1565.84 T D F Y H S K R R L I F 191 C₇₄H₁₁₂N₂₂O₁₇ 1581.82 D F Y H S K R R L I F S 192 C₇₃H₁₁₀N₂20₁₇ 1567 79 F Y H S K R R L I F S K 193 C₇₅H₁₁₇N₂₃O₁₅ 1580.88 Y H S K R R L I F S K R 194 C₇₂H₁₂₀N₂₆O₁₅ 1589 89 H S K R R L I F S K R K 195 C₆₉H₁₂₃N₂₇O₁₄ 1554 89 S K R R L I F S K R K P 196 C₆₈H₁₂₃N₂₅O₁₄ 1514 86 A F Y H S K R R L I F S 197 C₇₂H₁₁₀N₂₂O₁₅ 1523 78 D A Y H S K R R L I F S 198 C₆₇H₁₀₆N₂₂O₁₇ 1491.70 D F A H S K R R L I F S 199 C₆₇H₁₀₆N₂₂O₁₆ 1475.70 D F Y A S K R R L I F S 200 C₇₀H₁₀₈N₂₀O₁₇ 1501 73 D F Y H A K R R L I F S 201 C₇₃H₁₁₀N₂₂O₁₆ 1551.79 D F Y H S A R R L I F S 202 C₇₀H₁₀₃N₂₁O₁₇ 1510.7 D F Y H S K A R L I F S 203 C₇₀H₁₀₃N₁₉O₁₇ 1482.68 D F Y H S K R A L I F S 204 C₇₀H₁₀₃N₁₉O₁₈ 1483 68 D F Y H S K R R A I F S 205 C₇₀H₁₀₄N₂₂O₁₇ 1525 71 D F Y H S K R R L A F S 206 C₇₀H₁₀₄N₂₂O₁₈ 1526 71 D F Y H S K R R L I A S 207 C₆₇H₁₀₆N₂₂O₁₇ 1491 70 D F Y H S K R R L I F A 208 C₇₃H₁₁₀N₂₂O₁₆ 1551 79 F Y H S K R R L I F S 209 C₆₉H₁₀₅N₂₁O₁₄ 1452 71 Y H S K R R L I F S 210 C₆₀H₉₆N₂₀O₁₃ 1305 53 H S K R R L I F S 211 C₅₁H₈₇N₁₉O₁₁ 1142 36 D F Y H S K R R L I F 212 C₇₀H₁₀₅N₂₁O₁₃ 1480.72 D F Y H S K R R L I 213 C₆₁H₉₆N₂₀O₁₄ 1333.54 D F Y H S K R R L 214 C₅₅H₈₅N₁₉O₁₃ 1220.38 D F Y H S K R R 215 C₄₉H₇₄N₁₈O₁₂ 1107 23 D F Y H S K R 216 C₄₁H₆₂N₁₄O₁₁  951 04 D F Y H S K 217 C₁₇H₃₀N₁₀O₁₀  794.85 F Y H S K R R L I F 218 C₆₆H₁₀₀N₂₀O₁₂ 1365 63 F Y H S K R R L I 219 C₅₇H₉₁N₁₉O₁₁ 1218 45 F Y H S K R R L 220 C₅₁H₈₀N₁₈O₁₀ 1105.3 F Y H S K R R 221 C₄₅H₆₉N₁₈O₁₀  992 14 F Y H S K R 222 C₃₉H₅₇N₁₃O₈  835 95 Y H S K R R L I F 223 C₅₇H₉₁N₁₉O₁₁ 1218 45 Y H S K R R L I 224 C₄₈H₈₂N₁₈O₁₀ 1071 28 Y H S K R R L 225 C₄₂H₇₁N₁₇O₉  958 12 Y H S K R R 226 C₃₆H₆₀N₁₆O₈  844.96 Y H S K R 227 C₃₀H₄₈N₁₂O₇  688 78 H S K R R L I F 228 C₄₈H₈₂N₁₈O₉ 1055.28 S K R R L I F 229 C₄₂H₇₅N₁₅O₈  918 14 K R R L I F  34 C₃₉H₇₀N₁₄O₆  831 06 H S K R R L I 230 C₃₉H₇₃N₁₇O₈  908 11 H S K R R L 231 C₃₃H₆₂N₁₆O₈  794 95 K R R L I F S K 232 C₄₈H₈₇N₁₇O₉ 1046.31 RP-HPLC^(c) Cyclin E/CDK2 Cyclin D1/CDK4 MS^(b) T_(r) Purity % % [M + H] (min) (%) IC₃₀(μM) Inhibition IC₃₀(μM) Inhibition 1530.3 11.0 55.7 — 35 — 30  1541.66 11.5 73.5 — 40 — 18 1569.5 12.2 93.5 — 35 — 11 1583.9 13.3 76.9 2.2 ± 0 4 85 15 ± 3  72 1569 7 12 8 92 7 4.5 ± 0 5 80 20 ± 2  70 1580.4 12 0 89 4  26 ± 6 2 70 41 ± 10 70 1592 0 11 2 90 3 17 6 ± 6.9  80 45 ± 10 60 1556.9 10.7 20.0 8 7 ± 2.5 90 34 ± 6  80 1518 7 10 7 87 9 46 ± 33 70 — 40 1526.0 12.7 92.3 11 ± 2  70 22 ± 4  72 1494.8 12.2 80.8 5.9 ± 0.4 85 37 ± 6  76 1482.2 12.5 91 2 5.3 ± 0.6 80 121 ± 31  56 1506.6 13.0 79.1 5.1 ± 0.5 80 73 ± 42 60 1554.2 12.9 97.8  0.04 ± 0.005 80  10 52 1512.9 13.8 91 6 12.9 ± 2.4  80 200 50 1485 7 13.3 72.9 — 25 — 30 1488 9 13.2 78.6 30 ± 8  70 — 30 1529.0 12 1 94.5 — 30 — 30 1527 9 12 0 94.8 14 ± 3  80 53 ± 20 61 1495 0 11 3 89.6 — 20 — 35 1551 1 13.1 93 0 5.4 ± 1 1 70  40 60 1450 2 12 6 83.2 6.8 ± 1.0 80 22 ± 5  70 1304.0 12.2 81 8 7.3 ± 0.8 80 20 ± 1  70 1141.0 12.0 94 4 3.4 ± 0 2 80 32 ± 6  65 1476.5 13.5 94.6  2 ± 0.2 75 13 ± 2  70 1331.2 12 1 89.0 — 35 — 20 1219.6 10.6 98.0 200 50 — 10 1106 9  9 8 96.4 — 40 — 10  950 8  9.6 89.8 200 50 — —  794 4  9.5 96.9 200 45 — 10 1362 6 13 3 85.5 5 8 ± 1  80 19 ± 3  70 1218 2  9 6 68 2 — 45 — 20 1104.5 10 4 86.9 >200 48 — 20  994.3 9.2 83.6 >200 45 — 20  838 2 8 9 92.4 — 20 — 10 1218 8 12.9 94.3 7 ± 2 80 16 ± 1  70 1072 4 10.9 82.5 >200 45 — 15  960.4  9.2 95.8 >200 30 — 10  847 4  7.4 87.2 — 40 — 10  691.2  7.0 66.9 — 25 — 10 1056.5 12.7 81.8 3.4 ± 1  80 21 ± 4  72  919.3  8.2 93.4 7.7 ± 0.5 80 54 72  823 2  7.4 99.1 1 1 ± 1.3 80 >200 72  909 0 10.6 86.7 — 35 — 10  797.5  8 7 89.9 >200 45 — — 1047 9 11.3 94.2 >200 60 — 20

Example 6 Specificity of Enzyme Inhibition

[0262] Effect of p21 (149-160) on CDK2-Cyclin E Induced Phosphorylation of Histone 1.

[0263] p21(149-160) was tested for inhibitory activity in a CDK2/cyclin E kinase assay with histone H1 as a substrate. The peptide was completely inactive as an inhibitor of CDK2/cyclin E-induced phosphorylation of histone H1 (FIG. 1).

[0264] One possible mechanism for inhibitory action is competition of the peptide with the substrate for binding to the kinase complex. If this is so, the peptide inhibitory activity will depend on the substrate concentration. The IC₅₀ of p21(149-160) in the presence of different concentrations of histone H1 was determined and p21(149-160) did not inhibit CDK2/cyclin E-induced phosphorylation of histone H1 at any of the substrate concentrations used. The most potent inhibitor of CDK2/cyclin E phosphorylation of GST-pRb (i.e. p21(149-160)Ser153Ala) was also tested for its ability to inhibit histone H1 phosphorylation induced by the same kinase complex (FIG. 2). Even this powerful inhibitor of the GST-pRb phosphorylation was completely inactive in inhibition of the phosphorylation of histone H1 induced by CDK2/cyclin E kinase complex. Full-length p21^(WAF1), on the other hand, inhibited strongly both the CDK2/cyclin E- and CDK4/cyclin D1-induced phosphorylation of GST-pRb and histone H1. The substrate-specific effect of p21 (149-4160) and its derivatives strongly suggests a mechanism of competitive binding of the peptide inhibitors and pRb to CDK2/cyclin E and CDK4/cyclin D1. The fact that p21(149-160) and its derivatives did not inhibit significantly the CDK1/cyclin B-induced phosphorylation of GST-pRb (see below) excludes a possibility of direct binding of the peptide to the substrate.

[0265] Effect of p21 (149-160) and its Derivatives on CDK1-Cyclin B Kinase Activity.

[0266] p21(149-160) and its derivatives were tested for ability to inhibit CDK1/cyclin B kinase activity in phosphorylating histone H1 or GST-pRb (Table 2). p21(149-160) and its Ala mutant p21(149-160) Ser153Ala did not have any significant effect on the CDK1/cyclin B-induced phosphorylation of histone H1. None of the tested peptides were able to inhibit significantly the CDK1/cyclin B-induced phosphorylation of GST-pRb and only the highest peptide concentrations used (200 μM) had a marginal inhibitory effect on CDK1/cyclin B kinase activity. When tested in the “pull-down” assay, immobilised p21 (149-160) was unable to precipitate cyclin B either as a monomer, or as a complex with CDK1. These data coincide with the very poor inhibitory activity of the original 20mer p21(141-160) peptide (Ball, K. L.; Lain, S.; F{dot over (a)}hraeus, R.; Smythe, C.; Lane, D. P. Curr. Biol. 1996, 7, 71-80) and the full-length p21^(WAF1) protein towards CDK1/cyclin B complex (Harper, J. W.; Elledge, S. J.; Keyomarsi, K.; Dynlacht, B.; Tsai, L. H.; Zhang, P.; Dobrowolski, S.; Bai, C.; Connell-Crowley, L.; Swindell, E.; et al. Molec. Biol. Cell 1995, 6, 387-400) and show that p21(149-160) and its derivatives retain the selectivity of the full-length protein. TABLE 2 Inhibition of CDK1-Cyclin B induced phosphorylation of Histone 1 and GST-Rb by p21 derived peptides. Histone GST-Rb Peptide Sequence IC50 [μM] IC50 [μM] P21 (149-160) DFYHSKRRLIFS (SEQ ID No. 13) >200  200 P21 (149-160)153A DFYHAKRRLIFS (SEQ ID No. 19)  200 >200 P21 (149-159) DFYHSKRRLIF (SEQ ID No. 1) Not tested >200

[0267] Effect of Purified P21^(WAF1) on CDK4-Cyclin D1 and CDK2-Cyclin E Kinase Activity

[0268] In order to evaluate the selectivity, specificity and potency of p21 (149-160) and its derivatives we compared their effect with the one of purified p21 on kinase activity of CDK2-Cyclin E and CDK4-Cyclin D1. The IC 50 values characterizing the inhibition of CDK4-Cyclin D1 and CDK2-Cyclin E induced phosphorylation of GST-Rb and CDK2-Cyclin E induced phosphorylation of Histone 1 by purified p21^(WAF1) are shown in Table 3. The IC 50 of the most active peptide—p21 (149-160) 153A for CDK2-Cyclin E induced phosphorylation of GST-Rb was 40 nM which is approximately 50 fold higher than the IC 50 value for p21^(WAF1). Purified p21 though, inhibited strongly the CDK2-Cyclin E induced phosphorylation of GST-Rb as well as Histone 1. The peptides derived from p21^(WAF1)-p21 (149-160) and p21 (149-160)153A peptides specifically inhibit the GST-Rb phosphorylation, but do not inhibit the Histone 1 phosphorylation induced by CDK2-Cyclin E. This substrate specific effect of p21 (149-160) and its derivatives strongly suggest a mechanism of competitive binding of the peptide inhibitors and Rb to CDK2-Cyclin E or CDK4-Cyclin D1. The fact that p21 (149-160) and its derivatives did not inhibit significantly the CDK1-Cyclin B induced phosphorylation of GST-Rb excludes a possibility for direct binding of the peptide to the substrate (see Table 2). TABLE 3 Inhibition of CDK4-Cyclin D1 and CDK2-Cyclin E kinase activity by purified p21^(WAF1) Inhibition by p21^(WAF1) Kinase complex Substrate IC50 [nM] CDK4-Cyclin D1 GST-Rb(772-928) 6.5 ± 0.8 CDK2-Cyclin E GST-Rb(772-928) 0.7 ± 0.2 CDK2-Cyclin E Histone 1 1.8 ± 0.4

Example 7 P21 (149-160) and its Derivatives do not Inhibit PKCα and ERK2 Kinase Activity In Vitro

[0269] To investigate further the specificity of p21 (149-160) and its derivatives we investigated the effect of the strongest inhibitors of CDK2-Cyclin E and CDK4-Cyclin D1 complexes on PKC α and ERK2 kinase activity (Table 4). None of the tested peptides (at concentrations up to 100 μM) had any inhibitory effect on PKCα phosphorylation of Histone 3 or ERK2 phosphorylation of Myelin Basic Protein. These results demonstrate further the selectivity of the inhibitory effect of the peptides derived from p21 C-terminus. TABLE 4 Effect of p21^(WAF1)-derived peptides on PKCα and ERK2 kinase activity (activities against CDK2/cyclin E and CDK4/cyclin D1 included for comparison) SEQ ID IC₅₀ (mM) IC₅₀ (mM) IC₅₀ (mM) IC₅₀ (mM) Peptide Sequence^(a) No. CDK4-D1 CDK2-E PKCa ERK2 p21 (148-159) TDFYHSKRRLIF 191 15 2.2 >100 >100 p21 (149-160) DFYHSKRRLIFS 192 20 4.5 >100 >100 p21 (149-160)S153A DFYHAKRRLIFS 201 10 0.04 >100 >100 p21 (149-159) DFYHSKRRLIF 212 13 2 >100 >100 p21 (150-159) FYHSKRRLIF 218 19 5.8 >100 >100 p21 (151-159) YHSKRRLIF 223 16 7 >100 >100 p21 (152-159) HSKRRLIF 228 21 3.4 >100 >100

Example 8 P21 (149-159) Binds to the Cyclin, but Does not Bind to the CDK Sub-Unit of CDK/Cyclin Complex. Binding of the Peptide to the Cyclin Does not Disrupt the Complex

[0270] A biotinylated version of p21(149-159) was used in “pull-down” experiments with the purified CDK sub-units, CDK2, CDK4, cyclin A, cyclin D1, and with the complexes of CDK2/cyclin A or CDK4/cyclin D1 kinases, in order to determine the binding partner of the peptide. The biotinylated peptide was pre-immobilised on streptavidin-agarose beads. FIG. 3 shows the profiles of the “pulled down” proteins, after SDS-PAGE, Western blotting and immunodetection. It was found that p21(149-159) bound to cyclin A and cyclin D1, but failed to interact with CDK2 or CDK4 in the absence of their respective cyclin partners. Both CDK2 and CDK4 were “pulled down”, however, with biotinylated p21(149-159)-streptavidin-agarose beads when they were in a complex with cyclin A or cyclin D1, respectively. Similar results were obtained with cyclin E and CDK2/cyclin E complex. These results suggest that binding of biotinylated p21(149-160) to the cyclin subunit does not disrupt the CDK/cyclin complex. Such a method may be utilised either alone or together with a candidate substance to identify cyclin binding moities and/or inhibitors of cyclin-CDK interaction.

Example 9 Comparison Between Peptides, Containing ZRXL Substrate Recognition Motif

[0271] Adams et al., (1996) identified a motif—ZXRL (SEQ ID No. 233) which is present in many CDK2/Cyclin A (E) substrates-E2F family transcription factors and pRb family proteins; the same motif is present in p21 (N- and C-terminus), p27 and p57 kinase inhibitors (see FIG. 2 in Adams et al.). When the substrate recognition motif was mutated in p107 (Rb related protein) or E2F1 their phosphorylation by CDK2-Cyclin A was prevented (Adams et al., 1996).

[0272] Our p21 (149-160) SAR data clearly show though that two amino acids outside of ZXRL (SEQ ID No. 233) motif are very important for the kinase inhibitory activity of p21 (C-terminus) derived peptide—A153 (which increases the potency approximately 100 fold) and F159 (which is vital for the kinase inhibition). To evaluate the importance of these flanking the ZXRL motif regions we designed peptides, hybrids between p21 (152-159) and LDL motif (derived from E2F family transcription factors) or LFG motif (derived from p21 N-terminus, p27 and p57 kinase inhibitors), between p21 (16-23) and LIF motif (derived from p21 C-terminus) and between p21 (152-159)A153 and LFG motif. Their ability to inhibit CDK2/Cyclin E, CDK2/Cyclin A or CDK4/Cyclin D1 phosphorylation of pRb was compared with the one of the original peptides derived form p21-N and C-terminus, p27, E2F1 and p107 (Table 5).

[0273] The main results as presented in Table 5 below, are:

[0274] 1. All peptides inhibited CDK2-Cyclin and were much less potent toward CDK4/Cyclin D1 kinase activity.

[0275] 2. CDK2/Cyclin A and CDK2/Cyclin E were inhibited with similar potency by the 8-mers with the exception of HAKRRLIF (SEQ ID No. 42) and KAURRLIF (SEQ ID No. 234) which were 10 fold more potent toward CDK2/Cyclin A than to CDK2/Cyclin E kinase activity.

[0276] 3. In the context of eight amino acid peptides alanine substitution of Ser153 led to significant increase of the kinase inhibitory potency of p21 (152-159)-100, 10 and 4 fold toward CDK2/Cyclin A, CDK2/Cyclin E and CDK4/Cyclin D1 phosphorylation of pRb respectively.

[0277] 4. The most potent inhibitors of pRb phosphorylation contain Ala on the second position and LIF motif; they are followed by the peptides containing Ala on the second position and LFG motif (with the exception of the p27 derived peptide which contain Gln instead of Arg on the 5^(th) position), Ser and LFG, and Ser and LIF containing peptides. The least potent were LDL containing peptides.

[0278] These results manifest the importance of Ala and LIF motif for the kinase inhibitory potency of the peptides.

[0279] Competitive Binding of Peptides, Containing Different Motifs (LIF, LFG, LDL) to Cyclin A or Cyclin D1.

[0280] The next important question was if these peptides share the same kinase inhibitory mechanism (bind to the same Cyclin docking site). To answer this question we developed a competitive binding assay where the influence of the 8-mers on Cyclin A (D1)-p21 (149-159) binding was studied (See Materials and Methods for more details).

[0281] The results from Cyclin D1 competitive binding assay are summarized on Table 6. For easy comparison, the data for CDK4/Cyclin D1 kinase inhibitory activity of the peptides are given in the same table. TABLE 5 Kinase inhibitory activity of LDL, LIF and LFG containing peptides, derived from E2F, p107, p21 N- and C-terminus and p27. Competitive Kinase Inhibition binding^(b) Cyclin Cyclin Cyclin Cyclin A/CDK2 E/CDK2 D1/CDK4 D1/CDK6 Cyclin Cyclin Seq % % % % A D1 ID IC₅₀ Inhi- IC₅₀ Inhi- IC₅₀ Inhi- IC₅₀ Inhi- IC₅₀ IC₅₀ Peptide Sequence^(a) No. (μM) bition (μM) bition (μM) bition (μM) bition (μM) (μM) P21 C-terminus HSKRRLIF 228 3.4 80 3.4 80 21 72 n/d n/d N/d 48 P21 C-terminus HAKRRLIF 36 0.021 88 0.35 81 6 82 5.8 100 0.3 13 (S153A) P21 C-terminius-LFG HSKRRLFG 235 1.4 78 1.6 82 n/a 42 n/d n/d 4.4 >200 hybrid P21 C-terminus-LDL HSKRRLDL 236 5.4 78 39 74 n/a 24 n/d n/d 5.8 >200 hybrid P21 C-terminus HAKRRLFG 237 0.67 78 0.9 82 30 70 n/d n/d 0.35 33 (S153A) LFG E2F1 PVKRRLDL 238 1.2 80 2.1 74 99 58 n/d n/d 1.2 >100 P27 SAURNLFG 41 6.1 80 2 82 n/a 46 n/d n/d 3.8 >200 P107 SAKRRLFG 239 0.73 75 0.5 86 17 78 n/d n/d 0.51 24 P21 N-terminus KAURRLFG 240 0.54 80 0.074 86 42 66 n/d n/d 0.75 134 P21 N-terminus- KAURRLIF 234 0.062 70 1.2 78 13 83 n/d n/d 0.3 20 LIF hybrid

[0282] We have demonstrated a very good agreement between the CDK4/Cyclin D1 kinase inhibition and Cyclin D1 competitive binding capabilities of the tested peptides. The highest potency to inhibit CDK4/Cyclin D1 phosphorylation of pRb and to compete with Biotinylated p21-(149-159) for binding to Cyclin D1 has HAKRRLIF (SEQ ID NO. 42) peptide. These results suggest a mode of kinase inhibition via binding to the cyclin and coincide well with our previous results from ‘pull down’ experiments showing that the p21 (C-terminus) peptides bind to the Cyclins but not to the CDKs.

[0283] Thus, peptides containing the LDL motif (HSKRRLDL (SEQ ID NO. 236) and PVKRRLDL(SEQ ID NO. 237) were not able to inhibit CDK4/Cyclin D1 or to compete with Biotin-DFYHSKRRLIF (SEQ ID NO. 1) for binding to Cyclin D1. However, peptides, containing LFG motif and Ala on second position were able to inhibit CDK4/Cyclin D1 and to compete with Biotin-DFYHSKRRLIF (SEQ ID NO. 1) for binding to Cyclin D1. The only exception of this rule is p27 derived peptide —SAURNLFG (SEQ ID NO. 41), where one of the important Arg residues is replaced with Asn. These results suggest that LFG and LIF peptides bind to the same site of Cyclin D1.

[0284] The results for Cyclin A competitive binding and CDK2/Cyclin A kinase inhibition of the peptides, containing LIF, LFG and LDL motifs are also shown in Table 5. There is a very good correlation between the CDK2/Cyclin A inhibition and Cyclin A binding capabilities of the tested peptides. The most potent inhibitor and strongest binding competitor was HAKRRLIF (SEQ ID NO. 36) peptide.

[0285] Specificity and Selectivity of HAKRRLIF (SEQ ID NO. 42) Kinase Inhibitory Activity.

[0286] Similarly to p21 (149-160) its derivative p21 (152-159)S153A was not able to inhibit Histone phosphorylation by CDK2/Cyclin A(E) complexes (data not shown). HAKRRLIF (SEQ ID NO. 42) was not effective as an inhibitor in CDK1/Cyclin B in vitro kinase assay with Histone or Rb as substrates. HAKRRLIF (SEQ ID NO. 42) did not inhibit PKCα induced phosphorylation of Histones.

[0287] Thus, we have defined a 8-amino acid peptide derived for p21 (C-terminus) with a single point mutation—S153A which has significantly higher kinase inhibitory activity than the original sequence. HAKRRLIF (SEQ ID NO. 42) inhibited most strongly CDK2/Cyclin A phosphorylation of pRb—with IC 50 of 20 nM. The inhibitory activity of the peptide correlates with its ability to bind the cyclin sub-unit. HAKRRLIF (SEQ ID NO. 42) is very selective and specific kinase inhibitor—it inhibits specificly only the pRb phosphorylation activity of G1 CDK/Cyclins and does not inhibit the mitotic CDK/Cyclins-CDK1/Cyclin B (or A), or PKC α. HAKRRLIF (SEQ ID NO. 42) has much higher specificity and selectivity than the full length p21 protein, which inhibits the Histone phosphorylation of CDK2/Cyclin kinases complexes and has some activity toward CDK1/Cyclin B.

Example 10 Competitive Cyclin A Binding of p21- and pRb(866-880)/pRb(870-877) Peptides

[0288] It has been shown (Adams, P. D.; Li, X.; Sellers, W. R.; Baker, K. B.; Leng, X.; Harper, J. W.; Taya, Y.; Kaelin, W. G. J. Molec. Cell Biol. 1999, 19, 1068-1080) that pRb contains a cyclin-binding motif in its C-terminus and that this motif is required for the protein's phosphorylation. To test if the mechanism of kinase inhibition of the p21(152-159)Ser153Ala peptide was indeed via competition with pRb for binding to the cyclin subunit, two synthetic pRb-derived peptides—pRb(866-880) and pRb(870-877), as well as the recombinant GST-pRb(772-928) used in the kinase assays (all containing the cyclin-binding motif) were compared with p21-derived peptides for binding to cyclin A. Table 6 shows that all three pRb-derived peptides were able to compete with the p21-derived peptides for binding to cyclin A, and vice versa. Interestingly, the longer synthetic peptide pRb(866-880) was less effective than its truncated version pRb(870-877). Probably the conformation of the latter peptide is more favourable for cyclin binding. This peptide contains a C-terminal Phe, which case was found considerably to enhance the kinase inhibitory and cyclin-binding activity in the case of the p21-derived peptides. TABLE 6 Competitive cyclin A binding of p21- and pRb(866-880)/pRb(870-877) peptides Competitive Cyclin A SEQ RP-HPLC^(b) Binding IC₅₀ (μM) ID MS^(a) t_(R) Purity immobilised immobilised Compound No. Formula M_(r) [M + R] (min) (%) pRb peptide^(c) P21 peptide^(d) H-His-Ala-Lys-Arg-Arg- 36 0.2 0.02 Leu-Ile-Phe-NH₂ H-Asp Phe Tyr His Ala Lys 242 C₇₀H₁₀₅N₂₃O₁₄ 1464.7 1466.0 15.8^(i) >95 0.1 n/d Arg Arg Leu Ile Phe NH₂ H-Ser-Asn-Pro-Pro-Lys-Pro- 243 C₇₀H₁₁₇N₂₃O₂₁ 1781.1 1780.0 18.1^(ii) >95 35 48 Leu-Lys-Lys-Leu-Arg-Phe- Asp-Ile-Glu-NH₂ H-Lys-Pro-Leu-Lys-Lys-Lue- 244 C₅₀H₁₀₅N₂₃O₈ 1028.3 1026.0 17.00^(iii) >95 0.6 24 Arg-Phe-NH₂ GST-pRb(772-298)^(e) n/d 9

Example 11 Competitive Binding of P21^(WAF1) and H-His-Ala-Lys-Arg-Arg-Leu-Ile-Phe-NH₂ (SEQ ID No. 36) to Cyclin A in the Presence and Absence of CDK2

[0289] p21^(WAF1) contains two cyclin-binding sites, one each in its N- and C-terminus [(p21(19-23) and p21(154-159)], as well as a CDK2-binding site [p21(46-65)]. The cyclin A-binding affinities of full-length p21^(WAF1) and the peptide containing only the C-terminal cyclin-binding motif were compared in the presence and absence of CDK2. This showed (Table 7) that recombinant p21^(WAF1) had ca. 27-fold lower affinity than H-His-Ala-Lys-Arg-Arg-Leu-Ile-Phe-NH₂ (SEQ ID NO. 36) for cyclin A alone. When cyclin A was pre-complexed with CDK2, on the other hand, the apparent binding affinity of p21^(WAF1) increased and was comparable to that of the octapeptide. The increased ability of p21^(WAF1) to compete with the octapeptide for binding to CDK2-complexed cyclin A is most probably due to the contribution of the CDK-binding motif present in the former. On the other hand, the presence of CDK2 slightly decreased the apparent binding affinity of H-His-Ala-Lys-Arg-Arg-Leu-Ile-Phe-NH₂ (SEQ ID NO. 36) for cyclin A, which could be due to some conformational changes of the substrate recognition site on the cyclin sub-unit upon binding of CDK2. TABLE 7 Competitive binding of p21^(WAF1) and H-His-Ala-Lys-Arg-Arg-Leu-Ile-Phe- NH₂ to cyclin A in the presence and absence of CDK2 SEQ Comptetitve ID binding Protein Test Ligand in Solution No. IC₅₀ (nM)^(a) Cyclin A H-His-Ala-Lys-Arg Arg-Leu-Ile Phe-NH₂ 36 14 Cyclin A human recombinant p21 ^(WAF1) 289 Cyclin A/CDK2 complex H-His Ala Lys-Arg-Arg-Leu-Ile-Phe-NH₂ 36 28 Cyclin A/CDK2 complex human recombinant p21 ^(WAF1) 11

Examples 12-22 Structure-Activity Relationships of the p21(152-159)Ser153Ala Peptide (H-His-Ala-Lys-Ary-Ary-Leu-Ile-Phe-NH₂) (SEQ ID NO. 36)

[0290] For the purposes of the following examples, the reference peptide of the invention has been taken as HAKRRLIF (SEQ ID NO. 42) i.e. a preferred peptide of the invention in accordance with the third aspect. As such, the relative activity is expressed against this peptide and all relative activities approaching (over about 0.7) or greater than unity indicate peptides that may be classified as preferred. The comments provided in these Examples are made with this comparator in mind. It should however be borne in mind that even a peptide having a relative activity of <0.1, remains within the scope of the present invention by virtue of still being active in the context of the invention, such variants are variants upon the first or second embodiments as described above.

Example 12 Sensitivity to Chiral Changes

[0291] Each residue in turn was substituted by its chiral antipode and the resulting peptide analogues were tested for both CDK2/cyclin A kinase inhibition and competitive cyclin A binding in the presence of immobilised p21(152-159)Ser153Ala peptide. It was found that inversion of configuration at the Ca atoms was only tolerated (in terms of retention of biological activity) at the peptide's termini. Thus His¹⁵² could be present as either the L- or D-amino acid without loss of potency. Some potency was lost for the corresponding change at position Ala¹⁵³. Lys¹⁵⁴-Ile¹⁵⁸ could not be substituted by the corresponding D-amino acids without near-complete loss of activity. Some activity was retained when Phe¹⁵⁹ was inverted. These results confirm the highly selective and specific binding mode of the lead peptide. The effects seen for the terminal residues probably reflect the fact that these residues are conformationally more flexible in solution than sequence-internal groups and can be brought into a productive binding mode upon binding.

Example 12 D-Amino Acid Substitutions Based on p21(152-159)Ser153Ala

[0292] SEQ RP-HPLC^(b) Relative activity ID MS^(a) t_(R) Purity Kinase Cyclin A Compound No. Formula M_(r) [M + H] (min) (%) Inhibition^(c) Binding^(d) H-His-Ala-Lys-Arg-Arg-Leu-Ile-Phe-NH₂ 36 C₄₈H₈₂N₂₁O₈ 1039.3 1 1 H-his-Ala-Lys-Arg-Arg-Leu-Ile Phe-NH₂ 183 C₄₈H₈₂N₂₁O₈ 1039.3 1039.1 15.4 96 1.7 0.9 H-His-ala-Lys-Arg-Arg-Leu-Ile Phe-NH₂ 246 C₄₈H₈₂N₂₁O₈ 1039.3 1042.5 15.3 98 0.3 0.6 H-His-Ala-lys-Arg-Arg-Leu-Ile Phe-NH₂ 247 C₄₈H₈₂N₂₁O₈ 1039.3 1042.9 15.6 100 <0.1 <0.1 H-His-Ala-Lys-arg-Arg-Leu-Ile Phe-NH₂ 248 C₄₈H₈₂N₂₁O₈ 1039.3 1041.6 15.2 99 <0.1 <0.1 H-His-Ala-Lys-Arg-arg-Leu-Ile Phe-NH₂ 249 C₇₀H₁₀₅N₂₁O₁₄ 1039.3 1041.1 15.2 99 <0.1 <0.1 H-His-Ala-Lys-Arg-Arg-leu-Ile Phe-NH₂ 82 C₈₂H₁₃₇N₂₃O₂₁ 1039.3 1041.0 17.6 100 <0.1 <0.1 H-His-Ala-Lys-Arg-Arg-Leu-ile Phe-NH₂ 250 C₅₀H₈₉N₁₅O₈ 1039.3 1040.5 18.1 100 <0.1 <0.1 H-His-Ala-Lys-Arg-Arg-Leu-Ile phe-NH₂ 251 C₄₈H₈₂N₂₁O₈ 1039.3 1039.7 17.1 100 0.1 0.2

[0293] Residue Substitutions

Example 13 His¹⁵²

[0294] This residue is comparatively insensitive to substitution. With the exception of Pya, all residue substitutions were either tolerated or even lead to enhanced binding and/or kinase inhibition potency. Furthermore, this residue can be truncated without significant loss in biological activity.

Example 13 Substitutions of His¹⁵² Residue in p21(152-159)Ser153Ala

[0295] SEQ RP-HPLC^(b) Relative activity ID MS^(a) t_(R) Purity Kinase Cyclin A Compound No. Formula M_(r) [M + H] (min) (%) Inhibition^(c) Binding^(d) H-His-Ala-Lys-Arg-Arg-Leu-Ile-Phe-NH₂ 36 1 1 H-Ala-Ala-Lys-Arg-Arg-Leu-Ile Phe-NH₂ 47 C₄₂H₈₀N₁₆O₈ 973.2 975.4 15.4 98 1.8 2.5 H-Ala-Lys-Arg-Arg-Leu-Ile-Phe-NH₂ 48 C₄₂H₇₅N₁₅O₇ 902.1 901.0 15.5 100 1 0.3 H-Pya-Ala-Lys-Arg-Arg-Leu-Ile Phe-NH₂ 49 C₄₂H₈₀N₁₅O₇ 1049.3 1050.6 15.4 98 <0.1 0.2 H-Thi-Ala-Lys-Arg-Arg-Leu-Ile Phe-NH₂ 50 C₄₉H₈₂N₁₆O₈S 1055.3 1055.5 16.3 100 2 0.2 H-Hse-Ala-Lys-Arg-Arg-Leu-Ile Phe-NH₂ 51 C₄₆H₈₂N₁₆O₉ 1003.3 1002.9 15.7 82 2 2 H-Phe-Ala-Lys-Arg-Arg-Leu-Ile Phe-NH₂ 52 C₅₁H₈₄N₁₆O₈ 1049.3 1052.3 16.3 100 3 1 H- Dab-Ala-Lys-Arg-Arg-Leu-Ile Phe-NH₂ 53 C₄₆H₈₃N₁₇O₈ 1002.3 1004.7 15.5 100 5 0.4

Example 14 Ala¹⁵³

[0296] This is the residue position where replacement of the native Ser with Ala resulted in a dramatic potency increase. Further potency enhancements are observed when short, straight-chain (Abu) or β-branched (Val, Bug) residues are introduced. Side chains containing more than three saturated carbon atoms in a straight chain are poorly tolerated.

Example 14 Substitutions of Ala¹⁵³ Residue in p21(152-159)Ser153Ala

[0297] Relative activity SEQ RP-HPLC^(b) Kinase ID MS^(a) Purity Inhibi- Cyclin A Compound No. Formula M_(r) [M + H] tg(min) (%) tion^(c) Binding^(d) H-His-Ala-Lys-Arg-Arg-Leu-Ile- 36 1 1 Phe-NH₂ H-His-Gly-Lys-Arg-Arg-Leu-Ile 54 C₄₇H₈₀N₁₈O₈ 1025.3 1026.8 15.2  98 0.1 0.1 Phe-NH₂ H-His-Abu-Lys-Arg-Arg-Leu-Ile 55 C₄₉H₈₄N₁₈O₈ 1053.3 1055.2 15.8 100 5 1.3 Phe-NH₂ H-His-Nva-Lys-Arg-Arg-Leu-Ile 56 C₅₀H₈₆N₁₈O₈ 1067.3 1069.1 16.0 100 <0.1 <0.1 Phe-NH₂ H-His-Bug-Lys-Arg-Arg-Leu-Ile 57 C₅₁H₈₃N₁₈O₈ 1081.4 1082.7 15.9 100 0.2 1.2 Phe-NH₂ H-His-Val-Lys-Arg-Arg-Leu-Ile 58 C₅₀H₈₆N₁₈O₈ 1067.3 1068.5 15.9 100 2 1.7 Phe-NH₂ H-His-Ile-Lys-Arg-Arg-Leu-Ile 59 C₅₁H₈₃N₁₈O₈ 1081.4 1081.9 16.1 100 0.5 0.2 Phe-NH₂ H-His-Phg-Lys-Arg-Arg-Leu-Ile 60 C₅₃H₈₄N₁₈O₈ 1101.4 1101.8 15.8,16.1^(c) 100 <0.1 <0.1 Phe-NH₂ H-His-Phe-Lys-Arg-Arg-Leu-Ile 61 C₅₄H₈₆N₁₈O₈ 1115.4 1115.8 16.5 100 0.5 0.2 Phe-NH₂

Example 15 Lys¹⁵⁴

[0298] Various non-isosteric replacements are tolerated to some extent. A significant potency increase is observed when the conservative Lys-to-Arg replacement is made.

Example 15 Substitutions of Lys¹⁵⁴ Residue in p21(152-159)Ser153Ala

[0299] Relative activity SEQ RP-HPLC^(b) Kinase ID MS^(a) Purity Inhibi- Cyclin A Compound No. Formula M_(r) [M + H] tg(min) (%) tion^(c) Binding^(d) H-His-Ala-Lys-Arg- Arg-Leu-Ile- 36 C₄₈H₈₂N₁₈O₈ 1 1 Phe-NH₂ H-His-Ala-Ala-Arg-Arg-Leu-Ile 62 C₄₅H₇₅N₁₇O₈ 982.2 983.6 15.6 99 <0.1 0.5 Phe-NH₂ H-His-Ala-Nle-Arg-Arg-Leu-Ile 63 C₄₈H₈₁N₁₇O₈ 1024.3 1022.9 16.8 97 0.3 0.2 Phe-NH₂ H-His-Ala-Abu-Arg-Arg-Leu-Ile 64 C₄₆H₇₇N₁₇O₈ 996.2 997.4 16.1 100  0.8 0.2 Phe-NH₂ H-His-Ala-Leu-Arg-Arg-Leu-Ile 65 C₄₈H₈₁N₁₇O₈ 1024.3 1025.5 16.8 97 0.1 1.4 Phe-NH₂ H-His-Ala-Arg-Arg-Arg-Leu-Ile 66 C₄₈H₈₂N₂₀O₈ 1067.3 1067.9 15.5 94 5.7 1.5 Phe-NH₂

Example 16 Arg¹⁵⁵

[0300] Only the conservative replacements with Cit and Lys are tolerated to some extent.

Example 16 Substitutions of Arg¹⁵⁵ Residue in p21(152-159)Ser153Ala

[0301] Relative activity SEQ RP-HPLC^(b) Kinase ID MS^(a) Purity Inhibi- Cyclin A Compound No. Formula M_(r) [M + H] tg(min) (%) tion^(c) Binding^(d) H-His-Ala-Lys-Arg-Arg-Leu-Ile- 36 C₄₈H₈₂N₁₈O₈ 1 1 Phe-NH₂ H-His-Ala-Lys-Ala-Arg-Leu-Ile 67 C₄₅H₇₅N₁₅O₈  954.2  954.9 16.0 95 <0.1 <0.1 Phe-NH₂ H-His-Ala-Lys-Cit-Arg-Leu-Ile 68 C₄₈H₈₁N₁₇O₉ 1040.3 1053.5 12.5 94 0.2 0.2 Phe-NH₂ H-His Ala-Lys- Hse-Arg-Leu-Ile 69 C₄₆H₇₇N₁₅O₉  984.2  985.9 15.8 100  <0.1 . <0.1 Phe-NH₂ H-His-Ala-Lys-Nle-Arg-Leu-Ile 70 C₄₈H₈₁N₁₅O₈  996.3  998.4 18.1 86 <0.1 <0.1 Phe-NH₂ H-His-Ala-Lys-Gln-Arg-Leu-Ile 71 C₄₇H₇₈N₁₆O₉ 1011.2 1012.9 15.6 98 <0.1 <0.1 Phe-NH₂ H-His-Ala-Lys-Lys-Arg-Leu-Ile 72 C₄₈H₈₂N₁₆O₈ 1011.3 1011.8 15.3 100  0.8 0.1 Phe-NH₂

Example 17 Arg¹⁵⁶

[0302] This residue was probed with replacements constraining the backbone dihedral angles in different ways (Ala, Pro, Aib, Sar), none of which were tolerated. Partially tolerated replacements with Cit or Ser indicate involvment in H-bonding.

Example 17 Substitutions of Arg¹⁵⁶ Residue in p21(152-159)Ser153Ala

[0303] Relative activity SEQ RP-HPLC^(b) Kinase ID MS^(a) Purity Inhibi- Cyclin A Compound No. Formula M_(r) [M + H] tg(min) (%) tion^(c) Binding^(d) H-His-Ala-Lys-Arg-Arg-Leu-Ile- 36 C₄₈H₈₂H₁₈O₈ 1 1 Phe-NH₂ H-His-Ala-Lys-Arg-Ala-Leu-Ile 74 C₄₅H₇₅H₁₅O₈ 954.2 954.9 16.1 100 <0.1 <0.1 Phe-NH₂ H-His-Ala-Lys-Arg- Asn-Leu-Ile 75 C₄₆H₇₆H₁₆O₉ 997.2 997.5 15.5  99 <0.1 <0.1 Phe-NH₂ H-His Ala-Lys-Arg-Pro-Leu-Ile 76 C₄₇H₇₇H₁₅O₈ 980.2 980.1 16.3 100 <0.1 <0.1 Phe-NH₂ H-His-Ala-Lys-Arg-Ser-Leu-Ile 77 C₄₅H₇₅H₁₅O₉ 970.2 970.2 16.1 100 0.7 0.2 Phe-NH₂ H-His-Ala-Lys-Arg-Aib-Leu-Ile 78 C₄₆H₇₇H₁₅O₈ 968.2 968.1 16.7  73 <0.1 <0.1 Phe-NH₂ H-His-Ala-Lys-Arg-Sar-Leu-Ile 79 C₄₅H₇₅H₁₅O₈ 954.2 955.4 16.5 100 <0.1 <0.1 Phe-NH₂ H-His-Ala-Lys-Arg-Cit-Leu-Ile 80 C₄₈H₈₁H₁₇O₉ 1040.3  1041.42   15.67 100 0.3 n/d Phe-NH₂

Example 18 Leu¹⁵⁷

[0304] This residue is very sensitive to replacement, even with nearly isosteric groups. Only the very conservative Leu-to-Ile replacement was tolerated somewhat.

Example 18 Substitutions of Leu¹⁵⁷ Residue in p21(152-159)Ser153Ala

[0305] Relative activity SEQ RP-HPLC^(b) Kinase ID MS^(a) Purity Inhibi- Cyclin A Compound No. Formula M_(r) [M + H] tg(min) (%) tion^(c) Binding^(d) H-His-Ala-Lys-Arg-Arg-Leu-Ile- 36 C₄₈H₈₂N₁₈O₈ 1 1 Phe-NH₂ H-His-Ala-Lys-Arg-Arg-Ala-Ile 81 C₄₈H₈₂N₁₈O₈  997.2  996.9 13.9 100 <0.1 <0.1 Phe-NH₂ H-His-Ala-Lys-Arg-Arg-leu-Ile 82 C₄₈H₈₂N₁₈O₈ 1039.3 1041.0 15.1 100 <0.1 <0.1 Phe-NH₂ H-His Ala-Lys-Arg-Arg-Ile-Ile 83 C₄₈H₈₂N₁₈O₈ 1039.3 1041.1 14.4 100 1.5 0.2 Phe-NH₂ H-His-Ala-Lys-Arg-Arg-Val-Ile 84 C₄₇H₈₀N₁₈O₈ 1025.3 1026.2 15.8 100 <0.1 <0.1 Phe-NH₂ H-His-Ala-Lys-Arg-Arg-Nle-Ile 85 C₄₈H₈₂N₁₈O₈ 1039.3 1040.2 15.8 100 <0.1 <0.1 Phe-NH₂ H-His-Ala-Lys-Arg-Arg-Nva-Ile 86 C₄₇H₈₀N₁₈O₈ 1025.3 1025.0 14.9 100 <0.1 <0.1 Phe-NH₂ H-His-Ala-Lys-Arg-Arg-Cha-Ile 87 C₅₁H₈₀N₁₈O₈ 1079.4 1079.2 17.5 100 <0.1 <0.1 Phe-NH₂ H-His-Ala-Lys-Arg-Arg-Phe-Ile 88 C₅₁H₈₀N₁₈O₈ 1073.3 1072.7 16.4 100 <0.1 <0.1 Phe-NH₂ H-His-Ala-Lys-Arg-Arg-1Nap-Ile 89 C₅₂H₈₂N₁₈O₈ 1123.4 1122.5 17.9 100 <0.1 <0.1 Phe-NH₂

Example 19 Ile¹⁵⁸

[0306] All substitutions with aliphatic and aromatic residues were tolerated to some extent. However, excision of the Ile residue abolished activity. These results indicate that this residue is not crucial for activity but may be important as a spacer group between the flanking Leu and Phe groups.

Example 19 Substitutions of Ile¹⁵⁸ Residue in p21(152-159)Ser153Ala

[0307] Relative activity SEQ RP-HPLC^(b) Kinase ID MS^(a) Purity Inhibi- Cyclin A Compound No. Formula M_(r) [M + H] tg(min) (%) tion^(c) Binding^(d) H-His-Ala-Lys-Arg- Arg-Leu-Ile- 36 C₄₈H₈₂N₁₈O₈ 1 1 Phe-NH₂ H-His-Ala-Lys-Arg-Arg-Leu-Ala- 90 C₄₅H₇₆N₁₈O₈  997.2  996.5 13.8 100 0.3 0.8 Phe-NH₂ H-His-Ala-Lys-Arg-Arg-Leu-Leu- 91 C₄₈H₈₂N₁₈O₈ 1039.3 1038.4 16.1 100 1.2 0.6 Phe-NH₂ H-His Ala-Lys-Arg-Arg-Leu-Val- 92 C₄₇H₈₀N₁₈O₈ 1025.3 1024.7 14.9 100 0.8 1.5 Phe-NH₂ H-His-Ala-Lys-Arg-Arg-Leu-Nle 93 C₄₈H₈₂N₁₈O₈ 1039.3 1040.3 16.3 100 0.4 0.3 Phe-NH₂ H-His-Ala-Lys-Arg-Arg-Leu-Nva 94 C₄₇H₈₀N₁₈O₈ 1025.3 1025.7 15.2 100 0.2 0.6 Phe-NH₂ H-His-Ala-Lys-Arg-Arg-Leu-Cha- 95 C₅₁H₈₆N₁₈O₈ 1079.4 1080.2 18.4 100 0.3 0.5 Phe-NH₂ H-His-Ala-Lys-Arg-Arg-Leu-Phe- 96 C₅₁H₈₀N₁₈O₈ 1073.3 1073.9 16.3 100 0.4 0.4 Phe-NH₂ H-His-Ala-Lys-Arg-Arg-Leu-1Nap- 97 C₅₅H₈₂N₁₈O₈ 1123.4 1122.9 18.2 100 0.5 0.5 Phe-NH₂ H-His-Ala-Lys-Arg-Arg-Leu-Phe-NH₂ 98 C₄₂H₇₁N₁₇O₇  926.1  924.8 13.8 100 <0.1 <0.1

Example 20 Phe¹⁵⁹

[0308] Only certain replacements with aromatic residues were tolerated. Notably pFPhe substitution resulted in an analogue with enhanced cyclin A-binding affinity.

Example 20 Substitutions of Phe¹⁵⁹ Residue in p21(152-159)Ser153Ala

[0309] Relative activity SEQ RP-HPLC^(b) Kinase ID MS^(a) Purity Inhibi- Cyclin A Compound No. Formula M_(r) [M + H] tg(min) (%) tion^(c) Binding^(d) H-His-Ala-Lys-Arg-Arg-Leu-Ile-  36 C₄₈H₈₂N₁₈O₈ 1 1 Phe-NH₂ H-His-Ala-Lys-Arg-Arg-Leu-Ile-  99 C₄₅H₈₈N₁₈O₈ 1005.3 1005.7 14.2  97 0.3 <0.1 Leu-NH₂ H-His-Ala-Lys-Arg-Arg-Leu-Ile- 100 C₄₈H₈₈N₁₈O₈ 1045.3 1045.5 16.9 100 <0.1 <0.1 Cha-NH₂ H-His-Ala-Lys-Arg-Arg-Leu-Ile- 101 C₄₉H₈₄N₁₈O₈ 1053.3 1052.8 15.8  96 <0.1 <0.1 Hof-NH₂ H-His-Ala-Lys-Arg-Arg-Leu-Ile- 102 C₄₈H₈₂N₁₈O₉ 1055.3 1054.6 13.3 100 0.3 0.2 Tyr-NH₂ H-His-Ala-Lys-Arg-Arg-Leu-Ile- 103 C₄₈H₈₁N₁₈O₉ 1057.3 1055.8 16.0 100 1 5 pFPhe-NH₂ H-His-Ala-Lys-Arg-Arg-Leu-Ile- 104 C₄₈H₈₁N₁₈O₈ 1057.3 1055.5 16.2 100 0.8 0.8 mFPhe-NH₂ H-His-Ala-Lys-Arg-Arg-Leu-Ile- 105 C₅₀H₈₃N₁₉O₈ 1078.3 1076.1 15.6  98 0.3 0.1 Trp-NH₂ H-His-Ala-Lys-Arg-Arg-Leu-Ile- 106 C₅₂H₈₄N₁₈O₈ 1089.3 1090.7 17.8 100 0.2 <0.1 1Nap-NH₂ H-His-Ala-Lys-Arg-Arg-Leu-Ile- 107 C₅₂H₈₄N₁₈O₈ 1083.3 1090.6 18.0 100 1.2 0.7 2Nap-NH₂ H-His-Ala-Lys-Arg-Arg-Leu-Ile- 108 C₄₅H₈₅N₁₉O₈ 1020.3 1021.5 11.6  66 <0.1 <0.1 Lys-NH₂ H-His-Ala-Lys-Arg-Arg-Leu-Ile- 109 C₄₉H₈₂N₁₈O₈ 1051.3 1052.3 15.6  91 0.3 <0.1 Tic-NH₂

Example 21 Substitutions of Phe¹⁵⁹ Residue in p21(152-159)Ser153Ala with Conformationally Defined Residues Fmoc-DL-threo-Pse-OH

[0310] To a solution of H-DL-threo-Pse-OH (1 g, 5.5 mmol) in 5% aq Na₂CO₃ (13 mL, 6 mmol), was added a solution of Fmoc-ONSu (1.7 g, 5 mmol) in THF (13 mL) over a period of 30 min. The mixture was stirred vigorously for 5 h. The solvent was evaporated to dryness in vacuo. The residual white solid was dissolved in H₂O (150 mL) and was washed with Et₂O (2×100 mL). The aqueous phase was acidified to pH 2 with 0.2 M aq HCl and a precipitate was obtained, which was extracted into EtOAc (2×100 mL). The combined extracts were washed with aq KHSO₄ and brine, dried (MgSO₄) and concentrated in vacuo to afford a crude product (1.32 g, 65%). This was dissolved in the minimum volume of EtOAc and dripped into vigorously stirred hexane to afford, after filtration and drying, the title compound (1.27 g, 63%). M.p. 107-108° C. TLC (EtOAc/AcOH, 99:1): R_(f)=0.27. RP-HPLC (Vydac 218TP54, 1 mL/min, 50-100% MeCN in 0.1% aq CF₃COOH over 20 min): t_(R)=7.2 min. ¹H-NMR (CDCl₃, 250 MHz), δ. 7.75 (2H, d, J=7.6 Hz, Fmoc aromatic H), 7.42-7.49 (2h, M, Fmoc aromatic H), 7.27-7.39 (9H, m, aromatic H), 5.67 (1H, d, J=9.0 Hz, NH), 5.45 (1H, d, J=2.4 Hz, C^(β)H), 4.68 (1H, dd, J=2.5, 8.8 Hz, C^(α)H), 4.27 (2H, m, Fmoc CH₂), 4.14 (1H, t, J=7.1 Hz, Fmoc CH); ¹³C-NMR (CDCl₃:d₆-DMSO, 62.9 MHz) δ. 172.28 (carbonyl C, acid), 155.98 (carbonyl C, urethane), 143.60, 143.54, 140.80 (quaternary C). 127.81, 127.28, 127.16, 126.71, 125.73, 124.99, 124.88, 119.53, 72.68 (CH), 66.45 (CH₂), 59.62, 46.69 (CH). HR-MS (FAB) calc. For C₂₄H₂₂NO₅ (MH⁺): 404.149798, found 404.148369.

Ac-DL-threo-Pse-OH

[0311] To a cold solution (5° C.) of H-DL-threo-Pse-OH (1 g, 5.5 mmol) and NaHCO₃ (1.85 g, 22.1 mmol) in H₂O (30 mL) was added Ac₂O (1.6 mL, 16.6 mmol) dropwise over a period of 15 min. The mixture was stirred vigorously at room temperature overnight. It was extracted with EtOAc (100 mL). The aqueous phase was acidified to pH 2 with aq KHSO₄ and the product was extracted into EtOAc (3×100 mL), and NaCl was added to aid the process. The organic extracts were combined and washed with aq KHSO₄, brine, dried (MgSO₄) and concentrated in vacuo to afford the title compound as a white solid (0.9 g, 73%). M.p. 142-143° C.; ES-MS⁺ m/z 224.2 (MH⁺), calc. 224.2; TLC (MeOH/CH₂Cl₂/AcOH, 20:79:1): R_(f)=0.41; RP-HPLC (Vydac 218TP54, 1 mL/min, 20-60% MeCN in 0.1% aq CF₃COOH over 25 min): t_(R)=3.6 min. ¹H-NMR (d₆-DMSO, 250 MHz) δ. 12.49-12.60 (1H, br. S, CO₂H), 7.99 (1H, d, J=9.1 Hz, NH), 7.07-7.39 (5H, m, ArH), 5.76-5.90 (1H, br. S, OH), 5.14 (1H, d, J=2.9 Hz, C^(β)H), 4.48 (1H, dd, J=3.0, 9.1 Hz, C^(α)H), 1.75 (3H, s, CH₃).

H-DL-threo-Pse-OMe.HCl

[0312] A stream of HCl gas was passed through a stirred suspension of H-DL-threo-Pse-OH (1 g, 5.5 mmol) in MeOH (30 mL) at 0° C. After ca. 30 min, dissolution was complete. Gas addition was continued for 2 h. The mixture was allowed to reach room temeperature, sealed, and left to stand overnight. Solvent was removed in vacuo to afford the title compound as an off-white solid (1.07 g, 83%). M.p. 154-156° C. (dec.); ES-MS⁺ m/z 195.9 (MH⁺), calc. 196.2; RP-HPLC (Vydac 218TP54, 1 mL/min, 20-60% MeCN in 0.1% aq CF₃COOH over 25 min): t_(R)=3.2 min; ¹H-NMR (d₆-DMSO, 250 MHz) δ. 8.54, (3H, br. S, NH₃ ⁺), 7.29-7.40 (5H, m, ArH), 5.03 (1H, d, J=5.6 Hz, C^(β)H), 4.16 (1H, m, C^(α)H), 3.61 (3H, s, CH₃).

Ac-DL-threo-Pse-OMe

[0313] To a vigorously stirred solution of H-DL-threo-Pse-OMe.HCl (0.5 g. 2.15 mmol) and NaOAc.(H₂O)₃ (1.17 g, 8.6 mmol) in H₂O (10 mL) at 5° C. was added Ac₂O (0.6 mL, 6.45 mmol) dropwise over 15 min. A white precipitate was formed within 10 min, and stirring was continued for 16 h at room temperature. The mixture was extracted with EtOAc (2×100 mL), and the organic phase was separated and washed with aq NaHCO₃ (2×50 mL) and brine (100 mL). The organic phase was dried (MgSO₄), and evaporated to dryness in vacuo to afford th title compound as a white solid (0.36 g, 71. %). M.p. 176-179° C.; ES-MS⁺ m/z 238.1 (MH⁺), calcd. 238.2; TLC (MeOH/CH₂Cl₂, 1:5): R_(f)=0.69; RP-HPLC (Vydac 218TP54, 1 mL/min, 20-50% MeCN in 0.1% aq CF₃COOH over 25 min): t_(R)=5.2 min. ¹H-NMR (d₆-DMSO, 250 MHz) δ. 8.20 (1H, d, J=8.8 Hz, NH), 7.20-7.39 (5H, m, ArH), 5.88 (1H, d, J=4.6 Hz, OH), 5.09 (1H, m, C^(β)H), 4.54 (1H, dd, J=3,7 8.8 Hz, C^(α)H), 3.61 (3H, s, CO₂CH₃), 1.76 (3H, s, NHCOCH₃).

Ac-L-threo-Pse-OH

[0314] To a suspension of Ac-DL-threo-Pse-OMe (100 mg, 0.42 mmol) in 0.05 M aq potassium phosphate buffer (14 mL) was added α-chymotrypsin (10 mg, 400 units). The pH was maintained at its initial value (pH 7-8) by the manual addition of 0.5 M phosphate buffer. The mixture was stirred vigorously overnight. It was extracted with EtOAc (3×50 mL) to remove Ac-D-threo-Pse-OMe. The aqueous phase was separated, acidified to pH 2 with 2 M aq HCl and extracted into EtOAc (3×100 mL). The organic extract was washed with brine, dried (MgSO₄), and evaporated to dryness in vacuo to afford a colourless oil (25 mg, 53%). The title compound was obtained as a white solid after lyophilisation from H₂O.

[0315] M.p. 160-163; [α]_(D) ²⁶+25.10 (c=1.0, AcOH); ES-MS⁺ m/z 224.1 (MH⁺), calcd. 224.2; RP-HPLC (Vydac 218TP54, 1 mL/min, 20-60% MeCN in 0.1% aq CF₃COOH over 25 min): t_(R)=3.6 min; ¹H-NMR (d₆-DMSO, 250 MHz) δ. 7.99 (1H, D, J=9.1 Hz, NH), 7.18-7.39 (5H, m, ArH), 5.14 (1H, d, J=3.0 Hz, C^(β)H), 4.48 (1H, dd, J=3.0, 9.1 Hz, C^(α)H), 1.74 (3H, s, CH₃).

Ac-D-threo-Pse-OMe

[0316] From the above reaction, the initial EtOAc extract (150 mL) was washed with aq NaHCO₃ (2×50 mL) and brine (2×50 mL), dried and evaporated to dryness in vacuo to afford the title compound as a white solid (48 mg, 96%). RP-HPLC (Vydac 218TP54, 1 mL/min, 20-60% MeCN in 0.1% aq CF₃COOH over 25 min): t_(R)=5.2 min).

Fmoc-L-threo-Pse-OH

[0317] A solution of Ac-L-threo-Pse-OH (40 mg, 0.18 mmol) in 6 M aqueous HCl (5 mL) was refluxed at 100° C. for 5 h. The solution was allowed to attain room temperature and the solvent was removed in vacuo. The residue was then dissolved in H₂O and lyophilised to afford H-L-threo-Pse-OH.HCl as a white foam. To this was added a solution of 5% aq Na₂CO₃ (0.5 mL, 0.22 mmol). Effervescence occurred, and the solution was adjusted to pH 9 using a further equivalent of 5% aq Na₂CO₃ (0.5 mL). A solution of Fmoc-ONSu (60 mg, 0.18 mmol) in THF (1 mL) was then added over a period of 10 min. The mixture was stirred vigorously at room temperature for a further 5 h. The solvent was evaporated in vacuo, and the resulting white solid was dissolved in H₂O (100 mL) and washed with diethyl ether (2×50 ml). The aqueous extract was acidified to pH 2 with 2 M aq HCl and a precipitate was obtained, which was extracted into EtOAc (3×60 ml). The organic extract was washed with aq KHSO₄ (100 mL) and brine (100 mL), dried (MgSO₄) and concentrated in vacuo to afford a crude product as a yellow oil (84 mg, quantitative). This was dissolved in the minimum volume of EtOAc and dripped into vigorously stirred hexane (120 mL) to afford, after filtration and drying, the title compound (47 mg, 65%) as a white crystalline solid. M.p. 127-129° C.; [α]_(D) ²²+27.90 (c=1.0, MeOH); ES-MS⁺ m/z 404.3 (MH⁺), calcd. 404.4; TLC (EtOAc/AcOH, 99:1); R_(f)=0.27; RP-HPLC (Vydac 218TP54, 1 mL/min, 50-100% MeCN in 0.1% aq CF₃COOH over 20 min): t_(R) 7.2 min; ¹H-NMR (d₆-DMSO, 250 MHz) δ. 7.88 (2H, d, J=7.5 Hz, Fmoc ArH), 7.67 (1H, d, J=7.2 Hz, Fmoc ArH), 7.63 (1H, d, J=7.5 Hz, Fmoc ArH), 7.31-7.43 (9H, m, ArH), 5.80 (1H, br. s, 0H), 5.16 (1H, d, J=3.3 Hz, C^(β)H), 4.29 (1H, dd, J=3.3, 9.4 Hz, C^(α)H), 4.01-4.16 (3H, m, Fmoc CH, CH₂). HR-MS (FAB) calcd. for C₂₄H₂₂NO₅ (MH⁺): 404.149798, found: 404.149850.

Fmoc-D-threo-Pse-OH

[0318] A solution of Ac-D-threo-Pse-OMe (150 mg, 0.63 mmol) in 6 M aq HCl (10 mL) was refluxed at 100° C. for 5 h. The solution was allowed to attain room temperature and the solvent removed in vacuo. The residual material was dissolved in H₂O and lyophilised to afford H-D-threo-Pse-OH.HCl as a pale yellow solid, which was immediately carried forward to the next step. In a similar manner to that described for the preparation of Fmoc-L-threo-Pse-OH, the crude title product was obtained as a yellow oil (229 mg, 90%). The crude product was dissolved in the minimum volume of EtOAc and dripped into vigorously stirred hexane (120 mL) to afford the title compound (174 mg, 68% overall) as an off-white crystalline solid. M.p.: 127-129° C.; [α]_(D) ²²-29.0° (c=1.0, methanol); APcI-MS⁺ m/z 404.0 (MH⁺), calcd. 404.4; TLC (EtOAc/AcOH, 99:1); R_(f)=0.27; RP-HPLC (Vydac218TP54, 1 ml/min, 50-100% MeCN in 0.1% aq CF₃COOH over 20 min): t_(R)=7.2 min; ¹H-NMR (d₆-DMSO, 250 MHz): δ. 7.88 (2H, d, J=7.2 Hz, Fmoc ArH), 7.68 (1H, d, J=7.2 Hz, Fmoc ArH), 7.63 (1H, d, J=7.5 Hz, Fmoc ArH), 7.22-7.41 (9H, m, ArH), 5.17 (1H, d, J=3.2 Hz, C^(β)H), 4.30 (1H, dd, J=3.3, 9.5 Hz, C^(α)H), 4.01-4.15 (3H, m, Fmoc CH, CH₂). HR-MS (FAB) calcd. for C₂₄H₂₂NO₅ (MH⁺): 404.149798, found: 404.149722.

[0319] Addition of Fmoc-Protected Amino Acids to 5-[4-(4-tolyl(chloro)methyl)phenoxy]pentanoyl amino-methylated Polystyrene

[0320] 5-[4-(4-Tolyl(chloro)methyl)phenoxy]pentanoyl aminomethylated polystyrene (0.064 mmol, theoretical loading 0.64 mmol g⁻¹; Atkinson, G. E.; Fischer, P. M.; Chan, W. C. J. Org. Chem. 2000, 65, 5048-5056) and Fmoc-protected amino acid (0.192 mmol) were suspended in CH₂Cl₂ (2 mL). Following the addition of Pr₂ ^(i)NEt (0.128 mmol), the resultant mixture was stirred gently at room temperature for 24 h. The resin was filtered, washed successively with DMF, CH₂Cl₂ and MeOH, and dried in vacuo.

[0321] Addition of Fmoc-Amino Alcohols to 5-[4-(4-tolyl(chloro)methyl)-phenoxy]pentanoyl Aminomethylated Polystyrene

[0322] To a mixture of 5-[4-(4-tolyl(chloro)methyl)phenoxy]pentanoyl aminomethylated polystyrene (0.06 mmol, theoretical loading 0.64 mmol g⁻¹;) and Fmoc-amino alcohol (0.19 mmol) in ClCH₂CH₂Cl (3 mL) and THF (1 mL) was added Pr₂ ^(i)NEt (0.10 mmol). The suspension was then gently agitated at room temperature for 48-72 h. The resin was filtered, washed successively with DMF, CH₂Cl₂ and MeOH, and dried in vacuo.

[0323] Synthesis of Peptides

[0324] Amino acyl or peptidyl resin (0.026 mmol) was placed in a reaction column, swollen in DMF for 18 h, and Fmoc-deprotected using 20% piperidine in DMF. The resin was then washed with DMF (10 min, 2.5 mL/min), and the sequence Boc-His(Trt)-Ala/Ser(Bu^(t))-Lys(Boc)-Arg(Pbf)-Arg(Pbf)-Leu-Ile (SEQ ID NO. 252) was assembled using an automated PepSynthesizer 9050 (MilliGen). Sequential acylation reactions were carried out at ambient temperature for 2 h using appropriate Fmoc-protected amino acids [Fmoc-Ile-OH. 141 mg; Fmoc-Leu-OH, 141 mg; Fmoc-Arg(Pbf)-OH, 260 mg; Fmoc-Lys(Boc)-OH, 187 mg; Fmoc-Ala-OH, 125 mg; Fmoc-Ser(t-Bu)-OH, 153 mg, Fmoc-His(Trt)-OH, 248 mg; 0.40 mmol] and carboxyl-activated using TBTU (128 mg, 0.40 mmol), HOBt (31 mg, 0.20 mmol) and Pr₂ ^(i)NEt (1.31 mL, 10% in DMF). Repetitive Fmoc-deprotection was achieved using 20% piperidine in DMF (6 min, 2.5 mL/min). After the final Fmoc-deprotection, the terminal amine group was Boc-protected with di-tert-butyl dicarbonate (87 mg, 0.40 mmol). The assembled N-Boc-protected peptidyl-resin was filtered, washed successively with DMF, CH₂Cl₂ and MeOH, and dried in vacuo. The resin product was suspended in a mixture of Pr₃ ^(i)SiH (0.1 mL) and H₂O (0.4 mL), followed by the addition of CF₃COOH (4.5 mL). The reaction mixture was gently stirred at room temperature for 2 h. The suspension was filtered, washed with CF₃COOH (5 mL) and the filtrate evaporated to dryness in vacuo. The residual material was triturated with Et₂O (15 mL) to yield a white solid. The desired synthetic peptide was lyophilised from H₂O overnight, and purified by preparative RP-HPLC.

[0325] Dehydration Reaction of Peptides

[0326] The N-Boc-protected peptidyl-resin containing a C-terminal Pse residue (0.02 mmol) was swelled in CH₂Cl₂ (0.5 mL) and THF (0.5 mL) in a 2-necked round-bottomed flask for 1 h under N₂. The solution was cooled to −78° C., and Et₃N (84 μL, 0.60 mmol) followed by SOCl₂ (10 μL, 0.08 mmol) were carefully added to the resin suspension. The mixture was stirred at −78° C. for 3.5 h, after which a further quantity of SOCl₂ (10 μL, 0.08 mmol) was added, and the stirred mixture was gradually warmed to −10° C. over a period of 2.5 h. The mixture was then stirred at 5° C. overnight. The resin was filtered, washed successively with DMF, CH₂Cl₂ and MeOH, and dried in vacuo. The resin product was suspended in a mixture of ethyl methyl sulfide (0.1 mL), Pr₃ ^(i)SiH (0.1 mL) and H₂O (0.4 mL), followed by the addition of CF₃COOH (4.4 mL). The suspension was gently stirred at ambient temperature for 2 h. The suspension was filtered, washed with CF₃COOH (5 mL) and the filtrate evaporated to dryness in vacuo. The residual material was then triturated with Et₂O to yield a yellow solid, which was lyophilised from water (5-10 mL) overnight and purified by preparative RP-HPLC.

[0327] Psa-Containing Peptides

[0328] A portion of the corresponding Pse-containing peptidyl resin (50 mg, 0.015 mmol, theoretical loading 0.293 mmol/g) was suspended in DMF (1 mL) and treated with Ac₂O (14 μL, 0.15 mmol), Pr₂ ^(i)NEt (5 μL, 0.02 mmol) and 4-(N,N-dimethylamino)pyridine (0.18 mg, 0.0015 mmol). The mixture was gently stirred at room temperature for 24 h. The resin was filtered, washed successively with DMF, CH₂Cl₂ and MeOH, and dried in vacuo. The resin product (50 mg) upon acidolytic treatment gave the crude product. Pure peptides were obtained after purification by preparative RP-HPLC. Relative activity SEQ RP-HPLC^(b) Kinase ID MS^(a) Purity Inhibi- Cyclin A Compound No. Formula M_(r) [M + H] tg(min) (%) tion^(c) Binding^(d) H-His-Ala-Lys-Arg-Arg-Leu-Ile-  36 C₄₈H₈₂N₁₈O₈ 1 1 Phe-NH₂ H-His-Ala-Lys-Arg-Arg-Leu-Ile 110 C₄₈H₈₂N₁₈O₉ 1055.9 1055.7 8.8 99 <0.1 0.2 L-Pse OH H-His-Ala-Lys-Arg-Arg-Leo-Ile 111 C₄₈H₈₂N₁₈O₉ 1055.9 1056.0 6.8 99 <0.1 0.1 D-Pse OH H-His-Ser-Lys-Arg-Arg-Leu-Ile 112 C₄₈H₈₂N₁₈O₁₀ 1071.3 1074.1 8.8 99 <0.1 <0.1 L-Pse OH H-His-Ser-Lys-Arg-Arg-Leu-Ile 113 C₄₈H₈₂N₁₈O₁₀ 1071.3 1073.0 6.8 99 <0.1 <0.1 D-Pse OH H-His-Ala-Lys-Arg-Arg-Leu-Ile 114 C₅₀H₈₄N₁₈O₁₀ 1097.3 1098.0 11.2  99 22 n/d L-Psa OH H-His-Ala-Lys-Arg-Arg-Leu-Ile 115 C₄₈H₈₄N₁₈O₁₀ 1097.3 1098.0 8.4 99 <0.1 n/d D-Psa OH H-His-Ser-Lys-Arg-Arg-Leu-Ile 116 C₅₀H₈₄N₁₈O₁₁ 1113.3 1114.9 10.8  99 <0.1 n/d L-Psa OH H-His-Ser-Lys-Arg-Arg-Leu-Ile 117 C₅₀H₈₄N₁₈O₁₁ 1113.3 1114.4 8 99 <0.1 n/d D-Psa OH H-His-Ala-Lys-Arg-Arg-Leu-Ile 118 C₄₈H₈₀N₁₈O₈ 1037.3 1038.4 8.8  99 3.3 0.2 Dhp OH H-His-Ser-Lys-Arg-Arg-Leu-Ile 119 C₄₈H₈₀N₁₈O₉ 1053.3 1054.6 8.8  99 0.4 n/d Dhp OH H-His-Ala-Lys-Arg-Arg-Leu-Ile 120 C₄₈H₈₃N₁₇O₈ 1026.3 1026.2 8.4  99 0.6 1.0 Pheol H-His-Ser-Lys-Arg-Arg-Leu-Ile 121 C₄₈H₈₃N₁₇O₉ 1042.3 1041.6 8.4  95 0.2 <0.1 Pheol

[0329] As is clear from the results presented above, the Phe¹⁵⁹ residue represents a key determinant in the p21(152-159) pharmacophore: its truncation abolishes activity and certain well-defined substitutions lead to enhanced potency. For this reason, further constriction of the Phe aromatic side chain may lock it into a bio-active conformation and further potency gains may be expected. Such conformational definition can be introduced in many different ways, e.g. through further substitution at C^(β) (as in Psa and Pse), introduction of unsaturation, particularly between C^(α) and C⁶² (as in Dhp), or by tethering of the aromatic system to the peptide backbone (C^(α) and NH), as e.g. in Tic (refer structures below).

Conformational Constriction of Phenylalanine

[0330] The resolution of β-hydroxy-α-amino acids by the action of proteases on a range of N-acyl methyl esters has been described (Chênevert, R.; Létourneau, M.; Thiboutot, S. Can. J. Chem. 1990, 68, 960-963). Using a similar method, we resolved N-acetyl-DL-threo-phenylserine methyl ester into enantiomers of high optical purity by α-chymotrypsin-mediated enzymatic hydrolysis. Chymotrypsin is specific for the 2S-enantiomer that is typically found in natural amino acids. N^(α)-Fmoc-L-threo-β-phenylserine and N^(α)-Fmoc-D-threo-β-phenylserine were then synthesised from the resolved enantiomers. In principle the same transformations are applicable in the case of erythro-phenylserine (refer FIG. 3 for stereochemistry of Pse). The protected amino acids were immobilised for standard solid-phase peptide synthesis on a novel synthesis linker (Atkinson, G. E.; Fischer, P. M.; Chan, W. C. J Org Chem. 2000, 65, 5048-5056). It was found that the hydroxyl function in Pse did not require temporary protection under the reaction conditions applied (Fischer, P. M.; Retson, K. V.; Tyler, M. I.; Howden, M. E. H. Intl. J. Peptide Protein Res. 1991, 38, 491-493).

[0331] The peptides with a C-terminal Dhp residue were obtained directly from the corresponding Pse-containing peptides Thus, protected peptidyl resins were treated with thionyl chloride and triethylamine (Stohlmeyer, M. M.; Tanaka, H.; Wandless, T. J. J. Am. Chem. Soc. 1999, 121, 6100-7101), which led to selective dehydration, via a cyclic sulphamidite intermediate, of the hydroxyethylene function in the Pse residue, thus furnishing upon release from the linker-resin the corresponding Dhp peptides. The nature of the reaction mechanism ensured that the intermediate cyclic sulphamidite formed from the threo-configuration of phenylserine, under basic conditions, eliminated SO₂ stereospecifically to yield the corresponding Z-Dhp isomer. Peptides H-His-Ser(Ala)-Lys-Arg-Arg-Leu-Ile-Dhp-OH (SEQ ID Nos. 118 and 119) were typically obtained in >30% purity when analysed by RP-HPLC and purified yields of 20-30%. Conversely, E-Dhp peptides would be obtained by analogous dehydration of erythro-Pse peptides. Protected Pse peptidyl resins were acetylated selectively at the free hydroxyl of the Pse residue to afford the corresponding O-acetylphenylserine (Psa) peptides.

[0332] Stereochemistry of 3-phenylserine. The cis (Z) and trans (E) isomers of dehydrophenylalanine are derived from threo- and erythro-phenylserine, respectively, by dehydration.

[0333] As far as biological activity is concerned, only the L-Pse/Psa p21(152-159) peptides were able to inhibit CDK2/cyclin A and/or to bind efficiently to cyclin A. Of these, H-His-Ala-Lys-Arg-Arg-Leu-Ile-[L-Psa]-OH (SEQ ID No. 114) was particularly potent. Both Z-Dhp peptides were biologically active; the Ala¹⁵³ analogue being more potent then the corresponding Ser¹⁵³ peptide. Furthermore, the terminal Phe residue in the p21(152-159) peptides was also replaced with phenylalaninol (Pheol). This substitution was comparatively well-tolerated, showing that the terminal peptide carboxamide (or carboxylate) is not essential in terms of biological activity.

Example 22 Multiple Substitutions in p21(152-159)Ser153Ala,Phe159 pFPhe

[0334] It was seen above that certain residue substitution in the p21(152-159) peptides were in fact tolerated, and, in some cases, led to increased potency. Some of these single substitutions were then combined in order to test if combinatorial modifications at various positions in the peptide would be additive and/or synergistic. The results suggest that some synergysm is obtained. E.g., combination of His152Ala and Phe159 pFPhe replacements yielded a peptide analogue with about 80-fold increased potency, whereas the same substitutions individually lead to 2.5- and 5-fold potency increase (in terms of cyclin A binding) only. Thus, combination of the His152Ala, Ser153Ala, and Phe159 pFPhe modifications permitted introduction of e.g. Lys154Abu, Arg155Gln, Arg156Cit, Arg156Ser, and Ile158Ala.

Example 22 Multiple Substitutions in p21(152-159)Ser153Ala,Phe159 pFPhe

[0335] Relative activity SEQ RP-HPLC^(b) Kinase ID MS^(a) Purity Inhibi- Cyclin A Compound No. Formula M_(r) [M + H] tg(min) (%) tion^(c) Binding^(d) H-His-Ala-Lys-Arg-Arg-Leu-Ile- 36  C₄₈H₈₂N₁₈O₈ 1 1 Phe-NH₂ H-Ala-Ala-Abu-Arg Arg Leu Ile- 122 C₄₇H₇₄N₁₅O₈F 948.15 948.16 18.88 99 60 n/d pFPhe-NH₂ H-Ala-Ala-Lys-Arg Arg Leu Ile- 123 C₄₂H₇₂N₁₃O₉ 922.11 922.11 1782 99 80 n/d pFPhe-NH₂ H-Ala-Ala-Lys-Arg Cit-Leu Ile- 124 C₄₀H₇₃N₁₆O₉F 992.2 922.2 16.94 99 10 n/d pFPhe-NH₂ H-Ala-Ala-Lys-Arg Arg Leu- 125 C₄₂H₇₃N₁₆O₈F 949.14 949.69 17.89 99 20 n/d Ala-pFPhe-NH₂ H-Ala-Ala-Abu-Arg Ser Leu Ile- 126 C₄₀H₆₇N₁₂O₉F 879.04 879.05 16.56 99 14 n/d pFPhe-NH₂ H-Ala-Ala-Lys- Gln-Arg-Leu Ile- 127 C₄₄H₇₅N₁₄O₉F 963.16 963.17 20.16 99  4 n/d pFPhe-NH₂ H-Ala-Lys-Arg-Arg-Leu Ile- 253 C₄₂H₇₅N₁₅O₇F 902.15 920.14 16.6 99  4 n/d pFPhe-NH₂

Example 23 Cyclic Peptides

[0336] Inspection of the appropriate contacts in the complex structure of cyclin A with a p27^(KIP1) fragment (Russo, A. A.; Jeffrey, P. D.; Patten, A. K.; Massague, J.; Pavletich, N. P. Nature 1996, 382, 325-31).; suggested a starting point for the design of such conformationally constrained peptides. Asn³¹ of the p27 sequence apparently participates in H-bonds not only to the cyclin groove, but also in intra-molecular H-bonding to Gly³⁴. It was therefore plausible that peptide analogues containing macrocyclic constraints approximating this situation may be bio-active. One such cyclic peptide, in which Asn was replaced with Lys and an amide bond patched between its E-amino group and the carboxyl group of Gly, was designed and modelled (FIG. 3).

[0337] While molecular modelling suggested that this approach may work, the question remained whether a synthetic peptide containing the same constraint would indeed be bio-active. For this reason a convenient synthetic route based on an alkanesulfonamide safety-catch linker (Backes, B. J.; Ellman, J. A. J. Org. Chem. 1999, 64, 2322-2330) was developed for the synthesis of the desired ‘side chain-to-tail’ cyclic peptides as set out below;

[0338] In this method, the immobilised alkanesulfonamide linker is acylated with the desired Fmoc-amino acid, peptide chain assembly is then continued using standard solid-phase peptide synthesis methods. The diamino acid residue which is to participate in the prospective cyclic lactam bond is introduced in an orthogonally protected form, e.g. using an Fmoc-diamino acid derivative bearing a side-chain Mtt amino protecting group. After complete chain assembly, the sulfonamide linker is activated through alkylation with iodoacetonitrile. The Mtt protecting group is then removed under mild acidolytic conditions. Intramolecular attack of the liberated amino group on the activated acyl sulfonamide function then results in liberation of the protected cyclic peptide from the solid phase. It is isolated, fully deprotected using strong acidolysis, and purified. A similar approach has recently been reported for the synthesis of ‘head-to-tail’ cyclic peptides (Zhang, Z.; Van Aerschot, A.; Hendrix, C.; Busson, R.; David, F.; Sandra, P.; Herdewijn, P. Tetrahedron 2000, 56, 2513-2522). ‘Side chain-to-tail’ cyclic peptides can be obtained through various known methods, using either solid phase—(refer, e.g., Mihara, H.; Yamabe, S.; Niidome, T.; Aoyagi, H. Tetrahedron Lett. 1995, 36, 4837-4840) or solution methods (refer, e.g., He, J. X.; Cody, W. L.; Doherty, A. M. Lett.Peptide Sci. 1994, 1, 25-30)

[0339] Using the above method, the peptides 5,8-cyclo-[H-His-Ala-Lys-Arg-Lys-Leu-Phe-Gly] (SEQ ID NO. 173) and 5,8-cyclo-[H-His-Ala-Lys-Arg-Orn-Leu-Phe-Gly] (SEQ. ID NO. 174) were then synthesised and characterised. The results clearly show that the cyclic constraint introduced is relevant to the peptide's bioactive conformation. Whereas the analogue containing Lys in position 5 was approximately 2 orders of magnitude less potent than the lead peptide H-His-Ala-Lys-Arg-Arg-Leu-Ile-Phe-NH₂, (SEQ ID NO. 36) the corresponding Orn analogue was nearly equipotent with the lead peptide.

[0340] 5,8-cyclo-[H-His-Ala-Lys-Arg-Lys-Leu-Phe-Gly](1, n=3) (SEQ ID NO. 173)

[0341] Fmoc-Gly-OH (0.64 g, 2.16 mmol) and 4-sulfamylbutyryl aminomethylpolystyrene resin (Novabiochem; 0.50 g, nominally 0.54 mmol) were suspended in DMF (4.25 mL), and Pr^(i) ₂NEt (0.56 mL, 3.24 mmol) was added. The mixture was stirred for 20 min. After this time, it was cooled to −23° C., and PyBOP (1.13 g, 2.16 mmol) was added in one portion. Stirring was continued overnight, and the reaction was allowed to warm to room temperature during that period. The resin was then washed thoroughly with DMF, drained, and treated with 50% acetic anhydride in CH₂Cl₂ (10 mL) for 1 h. After completion, the resin was washed successively with CH₂Cl₂, DMF, and Et₂O, and was dried. The linear peptide sequence Boc-His(Boc)-Ala-Lys(Boc)-Arg(Pmc)-Lys(Mtt)-Leu-Phe-Gly (SEQ ID NO. 254) was then assembled using an ABI 433A peptide synthesiser, employing standard Fmoc protection strategy chemistry. The final peptidyl resin was washed successively with CH₂Cl₂, DMF, and Et₂O, and was dried. An aliquot (0.49 g) was swelled in NMP (4 mL) and treated with iodoacetonitrile (0.37 mL, 5.0 mmol) and Pr^(i) ₂NEt (0.24 mL 1.25 mmol) under N₂, for 24 h. After this time, the resin was washed thoroughly with NMP (4×5 min), DMF, CH₂Cl₂, and Et₂O, before drying. The Lys⁵ Mtt side-chain protecting group was then removed by treatment with 1.5% CF₃COOH, 3% MeOH in 1,2-dichloroethane (3×5 mL, 5 min each), and the resin was then washed with further 1,2-dichloroethane, followed by 20% Pr^(i) ₂NEt in CH₂Cl₂ and Et₂O. The resin was then dried in vacuo.

[0342] The activated and Lys⁵ side chain-deprotected peptidyl resin (100 mg) was swelled in 1,4-dioxane (2 mL; dried over sodium-benzophenone), and dimethylaminopyridine (10 mg) was added. The mixture was then heated at reflux for 14 h, followed by filtering of the resin, and washing with DMF (2×5 mL, 5 min). The combined filtrate and washings were evaporated, and the residue was treated with 2.5% Pr₃ ^(i)SiH in CF₃COOH solution for 1 h. The peptide product was collected by precipitation in ice-cold Et₂O, and after washing was dried and fractionated by preparative RP-HPLC (Vydac 218TP1022, 9 mL/min, 13-23% MeCN in 0.1% aq CF₃COOH over 60 min). Fractions containing pure cyclised peptide were pooled and lyophilised to afford title compound (2.2 mg, 2.34 μmol, 6.5% w.r.t. initial resin loading). Anal. RP-HPLC: t_(R)=15.4 min (Vydac 218TP54, 1 mL/min, 25° C., 13-23% MeCN in 0.1% aq CF₃COOH over 20 min), purity>99% (λ=214 nm). DE MALDI-TOF MS: [M+H]⁺=937.8, C₄₄H₇₁N₁₅O₈ requires 938.14 (positive mode, α-cyano-4-hydroxycinnamic acid matrix. The presence of the 5,8-cyclic structure was verified by inspection of appropriate through-space cotinectivities in the NMR ROESY spectrum of the peptide.

[0343] 5,8-cyclo-[H-His-Ala-Lys-Arg-Orn-Leu-Phe-Gly](1, n=2) (SEQ ID NO. 174)

[0344] This compound was prepared in a manner analogous to that described above except that residue position 5 was Om (Fmoc-Orn(Mtt)-OH was used during chain assembly). A portion of the resin (200 mg) was then treated as above, to give the pure title compound (6.3 mg, 6.81 μmol, 8.9% w.r.t. initial resin loading). Anal. RP-HPLC: t_(R)=14.09 min (Vydac 218TP54, 1 mL/min, 25° C., 15-25% MeCN in 0.1% aq CF₃COOH over 20 min), purity>99% (λ=214 nm). DE MALDI-TOF MS: [M+H]⁺=926.4, C₄₃H₆₉N₁₅O₈ requires 924.1 (positive mode, α-cyano-4-hydroxycinnamic acid matrix. The presence of the 5,8-cyclic structure was verified by inspection of appropriate through-space connectivities in the NMR ROESY spectrum of the peptide. SEQ [Cyclin A] Immobilised Relative Compound ID No. (μg/mL) Ligand^(a) IC₅₀](μM) Activity H-His-Ala-Lys-Arg-Arg-Leu-  36  5 HAKRRLIF .03 ± .01 1 Ile-Phe-NH₂ 5,8-cyclo-[H-His-Ala-Lys-Arg- 173  5 HAKRRLIF 11.1 ± 0.7  0.03 Lys-Leu-Phe-Gly] 5,8-cyclo-[H-His-Ala-Lys-Arg- 174  5 HAKRRLIF 0.7 ± 0.5 0.5 Orn-Leu-Phe-Gly] H-His-Ala-Lys-Arg-Arg-Leu-Ile-  36 10 HAKRRLIF  0.1 ± 0.05 1 Phe-NH₂ 5,8-cyclo-[H-His-Ala-Lys-Arg- 173 10 HAKRRLIF 16 ± 5  0.06 Lys-Leu-Phe-Gly] 5,8-cyclo-[H-His-Ala-Lys-Arg- 174 10 HAKRRLIF 0.4 ± 0.2 0.25 Orn-Leu-Phe-Gly] H-His-Ala-Lys-Arg-Arg-Leu-Ile-  36  5 DFYHSKRRLIFS 0.09 ± 0.02 1 Phe-NH₂ 5,8-cyclo-[H-His-Ala-Lys-Arg- 173  5 DFYHSKRRLIFS 8 ± 1 0.01 Lys-Leu-Phe-Gly] 5,8-cyclo-[H-His-Ala-Lys-Arg- 174  5 DFYHSKRRLIFS 0.3 ± 0.2 0.3 Orn-Leu-Phe-Gly] H-His-Ala-Lys-Arg-Arg-Leu-Ile-  36  10 DFYHSKRRLIFS 1.8 ± 0.9 1 Phe-NH₂ 5,8-cyclo-[H-His-Ala-Lys-Arg- 173  10 DFYHSKRRLIFS 22 ± 8  0.08 Lys-Leu-Phe-Gly] 5,8-cyclo-[H-His-Ala-Lys-Arg- 174  10 DFYHSKRRLIFS 6 ± 7 0.3 Orn-Leu-Phe-Gly]

Example 24 Further Truncated Peptides

[0345] The following truncated peptides were prepared and screened for competitive cyclin A binding in accordance with the methods described above. The results demonstrate that N-terminally truncated analogues of the 8mer p21-derived peptide H-His-Ala-Lys-Arg-Arg-Leu-Ile-Phe-NH₂ (SEQ ID NO. 36), and, to a lesser extent, the p27-derived peptide H-Ser-Ala-Abu-Arg-Arg-Asn-Leu-Phe-Gly-NH₂ (SEQ ID NO. 41), retain appreciable cyclin A binding capacity at least down to the C-terminal 4mer sequences.

Example 24 Further Truncated Peptides

[0346] Cyclin A Binding^(c) Maxi- SEQ mum ID MS^(a) RP-HPLC^(b) Purity IC₅₀ Inhibi- Compound No. Formula M_(r) [M + H] T_(R)(min) (%) (μM) tion (%) H-Arg-Leu-Ile-Phe-NH₂ 24 C₂₇H₄₆N₈ ^(O) ₄ 546.71 548.6 15.01^(iii) 99 — 50(at 100 (μM) H-Arg-Arg-Leu-Ile-Phe-NH₂ 25 C₃₃H₅₈N₁₂O₅ 702.9 704.7 13.35^(iii) 99 5 100 H-Lys-Arg-Arg-Leu-Ile-Phe-NH₂ 23 C₃₉H₇₀N₁₄O₆ 831.07 832.8 12.63^(iii) 99 5 100 H-Ala-Lys-Arg-Arg-Leu-Ile-Phe-NH₂ 27 C₄₂H₇₅N₁₅O₇ 902.15 903.9 12.82^(iii) 99 2 100 H-His-Ala-Lys-Arg-Arg-Leu-Ile-Phe-NH₂ 28 C₄₈H₈₂N₁₈O₈ 1039.3 1040.4  12.91^(iii) 99   0.3 100 H-ASn-Leu-Phe-Gly-NH₂ 29 C₂₁H₃₂N₆O₅ 448.52 449.6 18.14^(i) 99 — 80(at 100 (μM) H-Arg-Asn-Leu-Phe-Gly-NH₂ 30 C₂₇H₄₄N₁₀O₆ 604.71 605.2 17.17^(i) 99 — 20(at 100 (μM) H-Abu-Arg-Asn-Leu-Phe-Gly-NH₂ 31 C₃₁H₅₁N₁₁O₇ 689.81 690.9 12.87^(ii) 99 — — H-Ala-Abu-Arg-Asn-Leu-Phe-Gly-NH₂ 32 C₃₄H₅₆N₁₂O₆ 760.89 761.4 13.61^(ii) 99 25  90 H-Ser-Ala-Abu-Arg-Asn-Leu-Phe-Gly-NH₂ 33 C₃₇H₆₁N₁₃O₁₀ 847.97 849.1 14.90^(ii) 99 15 100

Example 25 Peptide Analogues of H-Ala-Ala-Lys-Arg-Arg-Leu-Ile-pFPhe-NH₂ (SEQ ID NO. 123)

[0347] RP-HPLC^(b) SEQ MS^(a) Purity Compound ID No. Formula M_(r) [M + H]⁺ tg(min) (%) H-Ala-Ala-Lys-Arg-Arg-Leu- Ile- pFPhe -NH₂ 123 C₄₅H₇₉FN₁₆O₈  991.2  991.1 12.45 90 H-Gly-Ala-Lys-Arg-Arg-Leu- Ile- pFPhe -NH₂ 129 C₄₄H₇₇FN₁₆O₈  977.2  976.4 15.9 94 H-Ala-Ala-Lys-hArg-Arg-Leu- Ile- pFPhe -NH₂ 130 C₄₆H₈₁FN₁₆O₈ 1005.3 1004.1 12.47 85 H-Ala-Ala-Lys-Ser-Arg-Leu- Ile- pFPhe -NH₂ 131 C₄₂H₇₂FN₁₃O₉  922.1  921.0 12.64 87 H-Ala-Ala-Lys-Hse-Arg-Leu- Ile- pFPhe -NH₂ 132 C₄₃H₇₄FN₁₃O₉  936.1  935.5 12.68 87 H-Ala-Ala-Lys-Arg-Lys-Leu- Ile- pFPhe -NH₂ 133 C₄₅H₇₉FN₁₄O₈  963.2  962.3 12.24 90 H-Ala-Ala-Lys-Arg-Orn-Leu- Ile- pFPhe -NH₂ 134 C₄₄H₇₇FN₁₄O₈  949.2  948.3 12.35 95 H-Ala-Ala-Lys-Arg-Gln-Leu- Ile- pFPhe -NH₂ 135 C₄₄H₇₅FN₁₄O₉  963.2  962.6 12.58 93 H-Ala-Ala-Lys-Arg-Hse-Leu- Ile- pFPhe -NH₂ 136 C₄₃H₇₄FN₁₃O₉  936.1  934.9 12.83 90 H-Ala-Ala-Lys-Arg-Thr-Leu- Ile- pFPhe -NH₂ 137 C₄₃H₇₄FN₁₃O₉  936.1  934.8 12.88 92 H-Ala-Ala-Lys-Arg-Nva-Leu- Ile- pFPhe -NH₂ 138 C₄₄H₇₆FN₁₃O₈  934.2  932.6 13.74 93 H-Ala-Ala-Lys-Arg-Arg-Phg- Ile- pFPhe -NH₂ 139 C₄₇H₇₅FN₁₆O₈  934.2 1009.8 11.42 90 H-Ala-Ala-Lys-Arg-Arg-Met- Ile- pFPhe -NH₂ 140 C₄₄H₇₇FN₁₆O₈ 1011.2 1009.2 12.04 80 H-Ala-Ala-Lys-Arg-Arg-Ala- Ile- pFPhe -NH₂ 141 C₄₂H₇₃FN₁₆O₈ 1009.3  948.1 11.43 82 H-Ala-Ala-Lys-Arg-Arg-Hof- Ile- pFPhe -NH₂ 142 C₄₉H₇₉FN₁₆O₈  949.1 1038.0 13.37 88 H-Ala-Ala-Lys-Arg-Arg-Leu- Ile- pFPhe -NH₂ 123 C₄₆H₈₁FN₁₆O₈ 1039.3 1003.1 13.2 86 H-Ala-Ala-Lys-Arg-Arg-alle- Ile- pFPhe -NH₂ 144 C₄₅H₇₉FN₁₆O₈ 1005.3  989.5 12.32 75 H-Ala-Ala-Lys-Arg-Arg-Leu- Gly- pFPhe -NH₂ 145 C₄₁H₇₁FN₁₆O₈  991.2  934.6 11.25 84 H-Ala-Ala-Lys-Arg-Arg-Leu- βAla- pFPhe -NH₂ 146 C₄₂H₇₃FN₁₆O₈  935.1  947.9 14.3 94 H-Ala-Ala-Lys-Arg-Arg-Leu- Pgh- pFPhe -NH₂ 147 C₄₇H₇₅FN₁₆O₈  949.1 1009.7 12.8,14.1 88 H-Ala-Ala-Lys-Arg-Arg-Leu- Aib- pFPhe -NH₂ 148 C₄₃H₇₅FN₁₆O₈ 1011.2  961.7 15.7 95 H-Ala-Ala-Lys-Arg-Arg-Leu- Sar- pFPhe -NH₂ 149 C₄₂H₇₃FN₁₆O₈  963.2  947.8 11.4 87 H-Ala-Ala-Lys-Arg-Arg-Leu- Pro- pFPhe -NH₂ 150 C₄₄H₇₅FN₁₆O₈  949.1  973.8 11.9 90 H-Ala-Ala-Lys-Arg-Arg-Leu- Bug- pFPhe -NH₂ 151 C₄₅H₇₉FN₁₆O₈  975.2  990.2 15.6 90 H-Ala-Ala-Lys-Arg-Arg-Leu- Ser- pFPhe -NH₂ 152 C₄₂H₇₃FN₁₆O₉  965.1  964.4 14.1 85 H-Ala-Ala-Lys-Arg-Arg-Leu- Asp- pFPhe -NH₂ 153 C₄₃H₇₃FN₁₆O₁₀  993.2  992.4 14.2 95 H-Ala-Ala-Lys-Arg-Arg-Leu- Asn- Phe -NH₂ 154 C₄₃H₇₄FN₁₇O₉  992.2  990.5 13.8 94 H-Ala-Ala-Lys-Arg-Arg-Leu- pFPhe- Phe -NH₂ 155 C₄₈H₇₇FN₁₆O₈ 1025.2 1024.1 16.8 94 H-Ala-Ala-Lys-Arg-Arg-Leu- diClPhe- Phe -NH₂ 156 C₄₈H₇₆Cl₂N₁₆O₈ 1076.1 1074.9 18.9 92 H-Ala-Ala-Lys-Arg-Arg-Leu- pClPhe- Phe -NH₂ 157 C₄₈H₇₇ClN₁₆O₈ 1041.7 1041.1 17.8 95 H-Ala-Ala-Lys-Arg-Arg-Leu- mClPhe- Phe -NH₂ 158 C₄₈H₇₇ClN₁₆O₈ 1041.7 1058.1 17.9 95 H-Ala-Ala-Lys-Arg-Arg-Leu- oClPhe- Phe -NH₂ 159 C₄₈H₇₇ClN₁₆O₈ 1041.7 1041.0 17.2 95 H-Ala-Ala-Lys-Arg-Arg-Leu- pIPhe- Phe -NH₂ 160 C₄₈H₇₇₁N₁₆O₈ 1133.1 1132.6 18.5 95 H-Ala-Ala-Lys-Arg-Arg-Leu- TyreMe- Phe -NH₂ 161 C₄₉H₈₉N₁₆O₈ 1037.3 1036.7 16.4 91 H-Ala-Ala-Lys-Arg-Arg-Leu- Thi- Phe -NH₂ 162 C₄₆H₇₆N₁₆O₈S 1013.3 1012.7 16.1 95 H-Ala-Ala-Lys-Arg-Arg-Leu- Pya- Phe -NH₂ 163 C₄₇H₇₇N₁₇O₈ 1008.2 1007.1 13.5 86 H-Ala-Ala-Lys-Arg-Arg-Leu- Ile- diClPhe -NH₂ 164 C₄₅H₇₈Cl₂N₁₆O₈ 1042.1 1005.8 18.6 91 H-Ala-Ala-Lys-Arg-Arg-Leu- Ile- pClPhe -NH₂ 165 C₄₅H₇₉ClN₁₆O₈ 1007.7 1004.2 17.3 88 H-Ala-Ala-Lys-Arg-Arg-Leu- Ile- mClPhe -NH₂ 166 C₄₅H₇₉ClN₁₆O₈ 1007.7 1006.8 17.3 88 H-Ala-Ala-Lys-Arg-Arg-Leu- Ile- oClPhe -NH₂ 167 C₄₅H₇₉ClN₁₆O₈ 1007.7 1007.0 16.5 84 H-Ala-Ala-Lys-Arg-Arg-Leu- Ile- Phg -NH₂ 168 C₄₄H₇₈N₁₆O₈  959.2  958.8 14.6,15.8^(c) 95 H-Ala-Ala-Lys-Arg-Arg-Leu- Ile- TyrMe -NH₂ 169 C₄₆H₈₂N₁₆O₉ 1003.3 1002.8 15.7 90 H-Ala-Ala-Lys-Arg-Arg-Leu- Ile- Thi -NH₂ 170 C₄₃H₇₈N₁₆O₈S  979.3  978.6 15.1 87 H-Ala-Ala-Lya-Arg-Arg-Leu- Ile- Pya -NH₂ 171 C₄₄H₇₉N₁₇O₈  974.2  973.7 11.5 90 H-Ala-Ala-Lys-Arg-Arg-Leu- Ile- Inc -NH₂ 172 C₄₅H₇₉FN₁₆O₈  971.2 (878.99)   16.1 95

Example 26 Peptide Analogues of H-Ala-Ala-Lys-Arg-Arg-Leu-Phe-Gly-NH₂ (SEQ ID NO. 255)

[0348] SEQ RP-HPLC^(b) ID MS^(a) Purity Compound No. Formula M_(r) [M + H]⁺ tg(min) (%) H-Ala-Ala-Lys-Arg-Arg-Leu- Phe-Gly-NH₂ 255 C₄₁H₇₂N₁₆O₈ 917.1 916.1 13.7 94 H-Ala-Ala-Lys-hArg-Arg-Leu- Phe-Gly-NH₂ 256 C₄₂H₇₄N₁₆O₈ 931.2 929.4 13.8 93 H-Ala-Ala-Lys-Ser-Arg-Leu- Phe-Gly-NH₂ 257 C₃₈H₆₅N₁₃O₉ 848.0 847.4 14.1 95 H-Ala-Ala-Lys-Hse-Arg-Leu- Phe-Gly-NH₂ 258 C₃₉H₆₇N₁₃O₉ 862.0 861.1 13.9 90 H-Ala-Ala-Lys-Arg-Lys-Leu- Phe-Gly-NH₂ 259 C₄₁H₇₂N₁₄O₈ 889.1 888.8 13.5 90 H-Ala-Ala-Lys-Arg-Orn-Leu- Phe-Gly-NH₂ 260 C₄₀H₇₀N₁₄O₈ 875.1 874.6 13.5 95 H-Ala-Ala-Lys-Arg-Gln-Leu- Phe-Gly-NH₂ 261 C₄₀H₆₈N₁₄O₉ 889.1 887.7 13.7 86 H-Ala-Ala-Lys-Arg-Hse-Leu- Phe-Gly-NH₂ 262 C₃₉H₆₇N₁₃O₉ 862.0 861.3 13.9 88 H-Ala-Ala-Lys-Arg-Thr-Leu- Phe-Gly-NH₂ 263 C₃₉H₆₇N₁₃O₉ 862.0 860.4 14.3 90 H-Ala-Ala-Lys-Arg-Nva-Leu- Phe-Gly-NH₂ 264 C₄₀H₆₉N₁₃O₈ 860.1 858.7 15.6 85 H-Ala-Ala-Lys-Arg-Arg-Met- Phe-Gly-NH₂ 265 C₄₀H₇₀N₁₆O₈ S 935.2 934.1 10.9 93 H-Ala-Ala-Lys-Arg-Arg-Ala- Phe-Gly-NH₂ 266 C₃₈H₆₆N₁₆O₈ 875.0 872.2 12.7 95 H-Ala-Ala-Lys-Arg-Arg-Hof- Phe-Gly-NH₂ 267 C₄₅H₇₂N₁₆O₈ 965.2 962.9 15.1 81 H-Ala-Ala-Lys-Arg-Arg-Hle- Phe-Gly-NH₂ 268 C₄₂H₇₄N₁₆O₈ 931.2 930.1 15.2 94 H-Ala-Ala-Lys-Arg-Arg-alle- Phe-Gly-NH₂ 269 C₄₁H₇₂N₁₆O₈ 917.1 915.9 13.2 95 H-Ala-Ala-Lys-Arg-Arg-Leu- Tic-Gly-NH₂ 270 C₄₂H₇₂N₁₆O₈ 929.1 928.3 13.7 93 H-Ala-Ala-Lys-Arg-Arg-Leu- Pgh-Gly-NH₂ 271 C₄₀H₇₀N₁₆O₈ 903.1 902.0 12.3,13.7^(c) 95 H-Ala-Ala-Lys-Arg-Arg-Leu- pFPhe-Gly-NH₂ 272 C₄₁H₇₁FN₁₆O₈ 935.1 933.7 14.3 95 H-Ala-Ala-Lys-Arg-Arg-Leu- plPhe-Gly-NH₂ 273 C₄₁H₇₁IN₁₆O₈ 1043.0  1041.3  16.4 92 H-Ala-Ala-Lys-Arg-Arg-Leu- Thi-Gly-NH₂ 274 C₃₉H₇₀N₁₆O₈S 923.2 920.8 13.2 96 H-Ala-Ala-Lys-Arg-Arg-Leu- Pya-Gly-NH₂ 275 C₄₀H₇₁N₁₇O₈ 918.1 915.1 9.3 90 H-Ala-Ala-Lys-Arg-Arg-Leu- diClPhe-Gly-NH₂ 276 C₄₁H₇₀Cl₂N₁₆O₈ 986.0 984.2 17 95 H-Ala-Ala-Lys-Arg-Arg-Leu- pClPhe-Gly-NH₂ 277 C₄₁H₇₁ClN₁₆O₈ 951.6 950.2 15.5 95 H-Ala-Ala-Lys-Arg-Arg-Leu- mClPhe-Gly-NH₂ 278 C₄₁H₇₁ClN₁₆O₈ 951.6 949.8 15.5 95 H-Ala-Ala-Lys-Arg-Arg-Leu- oClPhe-Gly-NH₂ 279 C₄₁H₇₁ClN₁₆O₈ 951.6 949.9 15 95 H-Ala-Ala-Lys-Arg-Arg-Leu- 1Nap-Gly-NH₂ 280 C₄₅H₇₄N₁₆O₈ 967.2 965.7 16.3 95 H-Ala-Ala-Lys-Arg-Arg-Leu- 2Nap-Gly-NH₂ 281 C₄₅H₇₄N₁₆O₈ 967.2 966.1 16.4 95 H-Ala-Ala-Lys-Arg-Arg-Leu- Inc-Gly-NH₂ 282 C₄₁H₇₀N₁₆O₈ 915.1 917.8 14.36 90 H-Ala-Ala-Lys-Arg-Arg-Leu- Phe-Asp-NH₂ 283 C₄₃H₇₄N₁₆O₁₀ 975.2 972.5 13.6 95 H-Ala-Ala-Lys-Arg-Arg-Leu- Phe-Glu-NH₂ 284 C₄₄H₇₆N₁₆O₁₀ 989.2 987.5 13.3 93 H-Ala-Ala-Lys-Arg-Arg-Leu- Phe-Ser-NH₂ 285 C₄₂H₇₄N₁₆O₉ 947.2 944.7 13.1 95 H-Ala-Ala-Lys-Arg-Arg-Leu- Phe-Asn-NH₂ 286 C₄₃H₇₅N₁₇O₉ 974.2 972.6 13.3 95 H-Ala-Ala-Lys-Arg-Arg-Leu- Phe-Gln-NH₂ 287 C₄₄H₇₇N₁₇O₉ 988.2 986.9 12.5 95 H-Ala-Ala-Lys-Arg-Arg-Leu- Phe-Lys-NH₂ 288 C₄₅H₈₁N₁₇O₈ 988.2 987.0 13.6 95

Example 27 Peptide Pentamers

[0349] Binding Kinase SEQ IC₅₀ IC₅₀ (μM) ID (μM) CyclinA/ Sequence No. Cyclin A CDK2 Ac- Arg Arg Leu Asn Phe NH₂ 378 8.15 37.2 Ac- Arg Arg Leu Asn pFF NH₂ 379 1.25 3.35 Ac- Arg Arg Leu Asn mClF NH₂ 380 4.1 17.85 Ac- Arg Arg Leu Ala Phe NH₂ 381 Ac- Arg Arg Leu Ala pFF NH₂ 382 3.7 10.45 Ac- Arg Arg Leu Ala mClF NH₂ 383 10.125 19.4 Ac- Arg Arg Leu Gly Phe NH₂ 384 Ac- Arg Arg Leu Gly pFF NH₂ 385 11.8 23.25 Ac- Arg Arg Leu Gly mClF NH₂ 386 25.45 29.75 Ac- Arg Arg Ile Asn Phe NH₂ 387 Ac- Arg Arg Ile Asn pFF NH₂ 388 3.85 8.3 Ac- Arg Arg Ile Asn mClF NH₂ 389 17.15 59.5 Ac- Arg Arg Ile Ala Phe NH₂ 390 Ac- Arg Arg Ile Ala pFF NH₂ 391 7.75 40.35 Ac- Arg Arg Ile Ala mClF NH₂ 392 26.15 >80 Ac- Arg Arg Ile Gly Phe NH₂ 393 Ac- Arg Arg Ile Gly pFF NH₂ 394 Ac- Arg Arg Val Gly mClF NH₂ 395 Ac- Arg Arg Val Asn Phe NH₂ 396 Ac- Arg Arg Val Asn pFF NH₂ 397 Ac- Arg Arg Val Asn mClF NH₂ 398 Ac- Arg Arg Val Ala Phe NH₂ 399 Ac- Arg Arg Val Ala pFF NH₂ 400 Ac- Arg Arg Val Ala mClF NH₂ 401 Ac- Arg Arg Val Gly Phe NH₂ 402 Ac- Arg Arg Val Gly pFF NH₂ 403 Ac- Arg Arg Val Gly mClF NH₂ 404 >100 >80 Ac- Arg Ser Leu Asn Phe NH₂ 405 Ac- Arg Ser Leu Asn pFF NH₂ 406 Ac- Arg Ser Leu Asn mClF NH₂ 407 109.45 89.7 Ac- Arg Ser Leu Ala Phe NH₂ 408 Ac- Arg Ser Leu Ala pFF NH₂ 409 Ac- Arg Ser Leu Ala mClF NH₂ 410 Ac- Arg Ser Leu Gly Phe NH₂ 411 Ac- Arg Ser Leu Gly pFF NH₂ 412 Ac- Arg Ser Leu Gly mCIF NH₂ 413 Ac- Arg Ser Ile Asn Phe NH₂ 414 Ac- Arg Ser Ile Asn pFF NH₂ 415 Ac- Arg Ser Ile Asn mClF NH₂ 416 Ac- Arg Ser Ile Ala Phe NH₂ 417 Ac- Arg Ser Ile Ala pFF NH₂ 418 Ac- Arg Ser Ile Ala mCIF NH₂ 419 Ac- Arg Ser Ile Gly Phe NH₂ 420 Ac- Arg Ser Ile Gly pFF NH₂ 421 Ac- Arg Ser Ile Gly mCIF NH₂ 422 Ac- Arg Ser Val Asn Phe NH₂ 423 Ac- Arg Ser Val Asn pFF NH₂ 424 Ac- Arg Ser Val Asn mClF NH₂ 425 Ac- Arg Ser Val Ala Phe NH₂ 426 Ac- Arg Ser Val Ala pFF NH₂ 427 Ac- Arg Ser Val Ala mClF NH₂ 428 Ac- Arg Ser Val Gly Phe NH₂ 429 Ac- Arg Ser Val Gly pFF NH₂ 430 Ac- Arg Ser Val Gly mClF NH₂ 431 Ac- Arg Lys Leu Asn Phe NH₂ 432 Ac- Arg Lys Leu Asn pFF NH₂ 433 Ac- Arg Lys Leu Asn mClF NH₂ 434 17.6 24.8 Ac- Arg Lys Leu Ala Phe NH₂ 435 Ac- Arg Lys Leu Ala pFF NH₂ 436 6.05 14.55 Ac- Arg Lys Leu Ala mCIF NH₂ 437 24.9 >80 Ac- Arg Lys Leu Gly Phe NH₂ 438 Ac- Arg Lys Leu Gly pFF NH₂ 439 15.05 >80 Ac- Arg Lys Leu Gly mCIF NH₂ 440 Ac- Arg Lys Ile Asn Phe NH₂ 441 Ac- Arg Lys Ile Asn pFF NH₂ 442 10.35 32.95 Ac- Arg Lys Ile Asn mCIF NH₂ 443 Ac- Arg Lys Ile Ala Phe NH₂ 444 Ac- Arg Lys Ile Ala pFF NH₂ 445 77.35 >80 Ac- Arg Lys Ile Ala mCIF NH₂ 446 Ac- Arg Lys Ile Gly Phe NH₂ 447 Ac- Arg Lys Ile Gly pFF NH₂ 448 Ac- Arg Lys Ile Gly mCIF NH₂ 449 Ac- Arg Lys Val Asn Phe NH₂ 450 Ac- Arg Lys Val Asn pFF NH₂ 451 Ac- Arg Lys Val Asn mCIF NH₂ 452 Ac- Arg Lys Val Ala Phe NH₂ 453 Ac- Arg Lys Val Ala pFF NH₂ 454 Ac- Arg Lys Val Ala mCIF NH₂ 455 Ac- Arg Lys Val Gly Phe NH₂ 456 Ac- Arg Lys Val Gly PFF NH₂ 457 Ac- Arg Lys Val Gly mCIF NH₂ 458 Ac- Arg Arg Leu Asn pFF NH₂ 295 0.72 1.55 Ac- Arg Arg Leu Asn pFF NH₂ 379 4.65 7.95 Ac- Arg Arg Ile Asn pFF NH₂ 304 1.25 1.45 Ac- Arg Arg Ile Asn pFF NH₂ 388 9.75 12.6 Ac- Arg Arg Leu Ile pFF NH₂ 375 1.55 7.85 Ac- Arg Arg Leu Ile pFF NH₂ 459 16.00 29.8 Ac- Arg Arg Leu Ala pFF NH₂ 298 1.00 3.15 Ac- Arg Arg Leu Ala pFF NH₂ 382 10.73 15.45

Example 28 Assays

[0350] Example of a Cyclin Affinity Capture Method for the Identification of Peptide Inhibitors

[0351] Peptides were synthesized as described above. Cyclin D1 was expressed in E coli BL21(DE3) using PET expression vector and purified from the inclusion bodies. After refolding Cyclin D1 was cross-linked on SulfoLink agarose support (PIERCE). CDK4-6×His was expressed in Sf9 insect cells infected with the appropriate baculovirus construct and purified by metal-affinity chromatography (Quiagen). GST-Rb (773-924) was expressed in E coli and purified on a Glutathione-Sepharose column according the manufacturers instructions (Pharmacia). CDK4/Cyclin D1 phosphorilation of Rb was determined by incorporation of radio-labeled phosphate in GST-Rb in 96-well format kinase assay. The phosphorylation reaction mixture consisted of 50 mM HEPES pH7.4, 20 mM MgCl₂, 5 mM EDTA, 2 mM DTT, 20 mM-glicerophosphate, 2 mM NaF, 1 mM Na₃VO₄, 0.5 g CDK4, 0.5 g Cyclin D1, 10 1 GST-Rb Sepharose beads, 100 M ATP and 0.2 Ci ³²P-ATP. The reaction was carried out for 30 min at 30 C at constant shaking. The GST-Rb-Sepharose beads were washed with 50 mM HEPES and 1 mM ATP and the radioactivity was measured on Scintillation counter (Topcount, HP)

[0352] Three Dimensional Models

[0353] As described in Example 4 above, a computer generated model of a preferred peptide of the present invention (HAKRRLIF) (SEQ ID NO. 42) complexed to cyclin A has been generated using AFFINITY (Molecular Simulations Inc.). A representation of this complex is shown in FIG. 4. Using the bond dimension analysis the following cyclin A amino acids have been determined as important in forming associations with this peptide: Cyclin A residues Major Intermediate Minor p21 residue Interaction Interaction Interaction H E223, E224 W217, V219, V221 G222, Y225, I281 S408, E411 A Y225 E223 K D284 E220, V279 R I213 A212, V215, L218 Q406, S408 R D283 I213, L214 M210, L253 L L253 G257 L218, I239, V256 I R250, Q254 F I206, R211 T207, L214 M200

[0354] These results demonstrate that the p21^(WAF1)-derived C-terminal peptides inhibit the phosphorylation of CDK substrates by binding to the cyclin regulatory subunit of the complex. Through the homology of this sequence with the sequences that have been determined crystallographically in complex with cyclins (Brown, N. R.; Noble, M. E.; Endicott, J. A.; Johnson, L. N. Nat. Cell Biol. 1999, 1, 438-443; Russo, A. A.; Jeffrey, P. D.; Patten, A. K.; Massague, J.; Pavletich, N. P. Nature 1996, 382, 325-31), as well as by virtue of our experimental results, we can conclude that the p21^(WAF1) peptide interacts with the same region of the protein as observed in these structures. The substrate recruitment site from these complexes consists mainly of residues of the α1 and α3 helices, which form a shallow groove on the surface, comprised predominantly of hydrophobic residues. These residues are largely conserved in the A, B, E and D1 cyclins. Analysis of the X-ray crystallographically determined structure of the ternary complex of p27^(KIP1), CDK2 and cyclin A gives considerable insight into the structural basis for the interactions of the p21^(WAF1) peptides examined here. In addition to the available experimentally derived information, a model of cyclin A-bound form of p21(152-159)Ser153Ala has been generated using computational docking procedures. These allow for the complex nature of protein—protein interactions to be delineated in terms of side-chain and backbone flexibility and using a routine employing full molecular mechanics description of non-bonded interactions. The generated model (FIG. 4) gives additional understanding of the molecular basis of the affinity of the peptide for the cyclin groove since it reveals that the residues that are intolerant to substitution and deletion make important contacts with the protein.

[0355] As with Examples 12-22, the following discussion relates to observations made in respect of the peptide HAKRRLIF (SEQ ID NO. 42) and all conclusions drawn in respect of potency increasing or decreasing are to be so interpreted. Two immediate conclusions can be drawn from the structure regarding the explanation of the functional significance of residues and which cannot be readily made from the available experimental data. The first is the rationale for the significant potency increase observed in the Ser153Ala substitution and the second is the accommodation of an aromatic residue in either position 7 or 8 of the cyclin binding motif (position 7 in conjunction with Gly at position 8). The basis for this can be ascertained by comparing the X-ray structure of the p27^(KIP1) ternary complex with the binary docked model structure. For the interaction of the LFG motif in the p27 structure, the Leu and Phe residues insert into the hydrophobic pocket formed by Met²¹⁰, Ile¹²³, Trp²¹⁷, and Leu²⁵³ provide the majority of the binding interaction of this region with the cyclin molecule. For the interactions of the LIF motif, the backbone torsion angles of the peptide at positions 6, 7 and 8 adjust in order to allow the Phe side chain to rotate into the hydrophobic pocket and form a high degree of complementarity with the hydrophobic pocket residue of the groove. The Ile side chain at position 7 (158 of p21) rotates out of the pocket to accommodate the Phe and no longer makes any hydrophobic contacts (see FIG. 5). The conformational changes that the peptide undergoes relative to the p27 structure in order to adapt the position 8 Phe residue into the hydrophobic pocket are quite marked. The comparison of the bound peptide structures in FIG. 5 illustrates how the turn structure on the NLFG (SEQ ID NO. 289) sequence in p27 which forms both intra- and inter-molecular hydrogen bonds is no longer present in the p21 peptide structure and is replaced by a more extended backbone conformation.

[0356] This observation explains the ability of the spacer residue between the Leu and Phe not only to be tolerated but also to increase affinity significantly as suggested by the observation that HAKRRLIF (SEQ ID NO. 42) is more potent than is the hybrid peptide HAKRRLFG (SEQ ID NO. 44). The ability of position 7 analogues including Ala to retain binding with cyclin A also supports this conclusion. The second observation and explanation that can be extracted from the model is the reason for the ability of the Ala replacement at position 153 dramatically to increase binding. This residue in the model forms hydrophobic contact with a second minor pocket which is made up by the second face of the Trp involved in the major pocket and two other residues. In the docked model, this second minor pocket is more pronounced and forms more complementary interactions with Ala than is observed in the crystal structure. It is apparent from this site that placement of the polar Ser residue in this hydrophobic environment would not be favoured and in fact would destabilise the binding interaction of the p21 peptide for the cyclin.

[0357] Further examination of the cyclin-bound p21 complex gives further indications of the nature of the residues that contribute to the affinity of the peptide to the recruitment site and that are different to those in the cyclin binding motif of p27. These include the His at 20, position 1 (Ser27 in p27), Lys at position 2 (Cys), and Arg at position 5 (Asn). The Ser to His change from p27 to p21 does not appear to be a critical one since both the Ala replacement peptide (p21(149-160)His 152Ala) and the truncated peptide minus the residue at position 1 are essentially equipotent. This result is consistent with the binding model since this residue does not form any contacts with the protein with the exception of an H-bond donation of the terminal amino group. By contrast of the Cys to Lys variant, functional data indicates that the Ala mutant undergoes a two-fold reduction in its ability to phosphorylate pRb. From the calculated model, Lys¹⁵⁴ forms an ion pair interaction with Asp²⁸⁴, thus, suggesting the basis for the potency decrease with this residue. Finally the Asn to Arg (156 in p21) change leads to a six-fold reduction in potency suggesting that the guanidino function of position 5 contributes to the binding interaction. Again the model indicates that this residue plays an important role in forming hydrogen bonds corresponding to those observed to the Asn residue in the p27 structure and thereby contributing to validation of the docked model. In addition, the recently published structure of a p 107 peptide bound to cyclin A verifies the model since the homologous Arg in this structure H-bonds to Asp²⁸³, an interaction which is also observed in the docked complex (Brown, N. R.; Noble, M. E.; Endicott, J. A.; Johnson, L. N. Nat. Cell Biol. 1999, 1, 438-443).

[0358] Other than those interactions identified as being unique to the peptides of the present invention, there are the residues that are conserved between p27 and the p21 C-terminally optimised peptides that form similar interactions to those observed in the experimentally derived structure. In particular, Arg¹⁵⁵, forms H-bonding and electrostatic interactions with Asp²¹⁶ and Glu²⁰⁰ and Leu¹⁵⁷ of the hydrophobic motif inserts into the pocket in a similar orientation to that observed in the crystal structure.

[0359] In summary, the model structure of the potent CDK2 and CDK4 inhibitor peptide H-His-Ala-Lys-Arg-Arg-Leu-Ile-Phe-NH₂ (SEQ ID NO. 36) in complex with CDK2/cyclin A gives considerable insight into the intermolecular interactions involved in cyclin binding and hence into blocking of substrate recruitment. In conjunction with kinase activity data for the series of p21 truncation and substitution analogues, this model clearly defines the sequence and structural requirements of the cyclin binding motif.

[0360] The pFPhe⁸ derivative of the peptide H-His-Ala-Lys-Arg-Arg-Leu-Ile-Phe-NH₂ (SEQ ID NO. 36) was found to possess increased activity in binding assays with cyclin A. Molecular modelling docking simulations performed with this analogue (FIG. 6) suggested that the pFPhe derivative inserts deeper into the hydrophobic pocket of the cyclin groove. This appears to result from rearrangement of the residues of the pocket forming more complementary interactions with the pFPhe residue and probably results from the change in charge distribution of the ring relative to the unsubstituted amino acid. This apparent gain in peptide-receptor affinity due to improved hydrophobic interactions of the pFPhe residue suggests that reduction of molecular mass through further N-terminal truncation will be possible without severe loss of biological activity.

REFERENCES

[0361] Valerio, R. M., Bray, A. M., Campbell, M., Dipasquale, A., Margellis, C., Rodda, S. J., Geysen, H. M., and Maeji, N.J. (1993) Int. J. Peptide Protein Res. 42,1-9.

[0362] Fields. G. B., and Noble, R. L. (1990) Int. J. Peptide Protein Res. 35, 161-214.

[0363] King, D. S., Fields, C. G., and Fields, G. B. (1990) Int. J. Peptide Protein Res. 36, 255-266.

[0364] Ball, K. L., Lain, S., F{dot over (a)}hraeus, R., Smythe, C., and Lane, D. P. (1996) Current Biol. 7, 71-80.

[0365] Harper, J W, Tsai L-H, Zhang, P, Dobrovolski C, Connel-Crowley, (1995) Cell, 6:387-400.

[0366] Holstein, M, Sidranski, D., Vogelstein, B., Harris, C. (1991) Science. 253:49-53.

[0367] Lane D P (1992) Nature 358:15-16.

[0368] Momand,. J., Zambetti, G. P., Olson, D. C., Levine, A. J. (1992) Cell, 69:1237-1245.

[0369] Deng, C., Zhang, P., Harper, J. W., Elledge S. J., Leder P. (1995) Cell, 82: 675-864.

[0370] Adams, P., Sellers, W., Sharma, S., Wu, A., Nalin, C., Kaelin W. (1996) Mol. and Cell. Biol. 16: 6623-6633.

[0371] Chen et al. Mol Cell Biol (1996) 16 4673-4682

[0372] Lin J. et al., Mol Cell Biol (1996) 16 1768-1793

[0373] Russo A A et al., Nature (1996) 382: 325-331

[0374] Chen, et al., (1995) Nature, 374: 386-388

[0375] Flores-Rozas, et al. (1994) Prof. Natl. Acad. Sci. U.S.A., 91: 8655-8659

[0376] Luo, et al. (1995) Nature, 375: 159-161

[0377] Nakanishi, et al. (1995b) J. Biol. Chem., 270: 17060-17063

[0378] Warbrick, et al. (1995) Curr. Biol., 5: 275-282

[0379] Waga, et al. (1994) Nature, 369: 574-578

[0380] The contents of all references, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference.

EQUIVALENTS

[0381] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

1 459 1 11 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 1 Asp Phe Tyr His Ser Lys Arg Arg Leu Ile Phe 1 5 10 2 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 2 Xaa Xaa Xaa Xaa Xaa Leu Xaa Phe 1 5 3 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 3 His Xaa Lys Arg Arg Leu Xaa Phe 1 5 4 13 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 4 Xaa Asp Phe Tyr His Ser Lys Arg Arg Leu Ile Phe Xaa 1 5 10 5 5 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 5 Arg Arg Leu Ile Phe 1 5 6 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 6 Lys Arg Arg Leu Ile Phe Ser Lys 1 5 7 8 PRT Artificial Sequence Description of Artificial Sequence E2F1 derived peptide 7 Pro Val Lys Arg Arg Leu Asp Leu 1 5 8 8 PRT Artificial Sequence Description of Artificial Sequence E2F1 derived peptide 8 Pro Val Lys Arg Arg Leu Phe Gly 1 5 9 4 PRT Artificial Sequence Description of Artificial Sequence E2F1 derived peptide 9 Arg Xaa Leu Phe 10 6 PRT Artificial Sequence Description of Artificial Sequence E2F1 derived peptide 10 Arg Arg Leu Phe Gly Glu 1 5 11 4 PRT Artificial Sequence Description of Artificial Sequence E2F1 derived peptide 11 Xaa Leu Xaa Phe 1 12 5 PRT Artificial Sequence Description of Artificial Sequence E2F1 derived peptide 12 Arg Xaa Leu Xaa Phe 1 5 13 12 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 13 Asp Phe Tyr His Ser Lys Arg Arg Leu Ile Phe Ser 1 5 10 14 12 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 14 Thr Asp Phe Tyr His Ser Lys Arg Arg Leu Ile Phe 1 5 10 15 12 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 15 Ala Phe Tyr His Ser Lys Arg Arg Leu Ile Phe Ser 1 5 10 16 12 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 16 Asp Ala Tyr His Ser Lys Arg Arg Leu Ile Phe Ser 1 5 10 17 12 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 17 Asp Phe Ala His Ser Lys Arg Arg Leu Ile Phe Ser 1 5 10 18 12 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 18 Asp Phe Tyr Ala Ser Lys Arg Arg Leu Ile Phe Ser 1 5 10 19 12 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 19 Asp Phe Tyr His Ala Lys Arg Arg Leu Ile Phe Ser 1 5 10 20 12 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 20 Asp Phe Tyr His Ser Ala Arg Arg Leu Ile Phe Ser 1 5 10 21 12 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 21 Asp Phe Tyr His Ser Lys Arg Ala Leu Ile Phe Ser 1 5 10 22 12 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 22 Asp Phe Tyr His Ser Lys Arg Arg Leu Ala Phe Ser 1 5 10 23 12 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 23 Asp Phe Tyr His Ser Lys Arg Arg Leu Ile Phe Ala 1 5 10 24 11 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 24 Phe Tyr His Ser Lys Arg Arg Leu Ile Phe Ser 1 5 10 25 10 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 25 Tyr His Ser Lys Arg Arg Leu Ile Phe Ser 1 5 10 26 9 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 26 His Ser Lys Arg Arg Leu Ile Phe Ser 1 5 27 10 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 27 Phe Tyr His Ser Lys Arg Arg Leu Ile Phe 1 5 10 28 9 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 28 Tyr His Ser Lys Arg Arg Leu Ile Phe 1 5 29 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 29 His Ser Lys Arg Arg Leu Ile Phe 1 5 30 7 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 30 Ser Lys Arg Arg Leu Ile Phe 1 5 31 6 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 31 Lys Arg Arg Leu Ile Phe 1 5 32 4 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 32 Arg Leu Ile Phe 1 33 5 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 33 Arg Arg Leu Ile Phe 1 5 34 6 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 34 Lys Arg Arg Leu Ile Phe 1 5 35 7 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 35 Ala Lys Arg Arg Leu Ile Phe 1 5 36 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 36 His Ala Lys Arg Arg Leu Ile Phe 1 5 37 4 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 37 Asn Leu Phe Gly 1 38 5 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 38 Arg Asn Leu Phe Gly 1 5 39 6 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 39 Xaa Arg Asn Leu Phe Gly 1 5 40 7 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 40 Ala Xaa Arg Asn Leu Phe Gly 1 5 41 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 41 Ser Ala Xaa Arg Asn Leu Phe Gly 1 5 42 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 42 His Ala Lys Arg Arg Leu Ile Phe 1 5 43 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 43 His Ser Lys Arg Arg Leu Phe Gly 1 5 44 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 44 His Ala Lys Arg Arg Leu Phe Gly 1 5 45 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 45 Lys Ala Cys Arg Arg Leu Phe Gly 1 5 46 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 46 Lys Ala Cys Arg Arg Leu Ile Phe 1 5 47 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 47 Ala Ala Lys Arg Arg Leu Ile Phe 1 5 48 7 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 48 Ala Lys Arg Arg Leu Ile Phe 1 5 49 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 49 Xaa Ala Lys Arg Arg Leu Ile Phe 1 5 50 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 50 Xaa Ala Lys Arg Arg Leu Ile Phe 1 5 51 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 51 Xaa Ala Lys Arg Arg Leu Ile Phe 1 5 52 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 52 Phe Ala Lys Arg Arg Leu Ile Phe 1 5 53 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 53 Xaa Ala Lys Arg Arg Leu Ile Phe 1 5 54 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 54 His Gly Lys Arg Arg Leu Ile Phe 1 5 55 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 55 His Xaa Lys Arg Arg Leu Ile Phe 1 5 56 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 56 His Xaa Lys Arg Arg Leu Ile Phe 1 5 57 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 57 His Xaa Lys Arg Arg Leu Ile Phe 1 5 58 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 58 His Val Lys Arg Arg Leu Ile Phe 1 5 59 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 59 His Ile Lys Arg Arg Leu Ile Phe 1 5 60 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 60 His Xaa Lys Arg Arg Leu Ile Phe 1 5 61 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 61 His Phe Lys Arg Arg Leu Ile Phe 1 5 62 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 62 His Ala Ala Arg Arg Leu Ile Phe 1 5 63 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 63 His Ala Xaa Arg Arg Leu Ile Phe 1 5 64 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 64 His Ala Xaa Arg Arg Leu Ile Phe 1 5 65 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 65 His Ala Leu Arg Arg Leu Ile Phe 1 5 66 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 66 His Ala Arg Arg Arg Leu Ile Phe 1 5 67 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 67 His Ala Lys Ala Arg Leu Ile Phe 1 5 68 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 68 His Ala Lys Xaa Arg Leu Ile Phe 1 5 69 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 69 His Ala Lys Xaa Arg Leu Ile Phe 1 5 70 8 PRT Artificial Sequence Synthesised with free amino terminus and as the C-terminal Carboxamide 70 His Ala Lys His Arg Leu Ile Phe 1 5 71 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 71 His Ala Lys Xaa Arg Leu Ile Phe 1 5 72 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 72 His Ala Lys Gln Arg Leu Ile Phe 1 5 73 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 73 His Ala Lys Lys Arg Leu Ile Phe 1 5 74 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 74 His Ala Lys Arg Ala Leu Ile Phe 1 5 75 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 75 His Ala Lys Arg Asn Leu Ile Phe 1 5 76 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 76 His Ala Lys Arg Pro Leu Ile Phe 1 5 77 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 77 His Ala Lys Arg Ser Leu Ile Phe 1 5 78 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 78 His Ala Lys Arg Xaa Leu Ile Phe 1 5 79 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 79 His Ala Lys Arg Xaa Leu Ile Phe 1 5 80 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 80 His Ala Lys Arg Xaa Leu Ile Phe 1 5 81 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 81 His Ala Lys Arg Arg Leu Ile Phe 1 5 82 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 82 His Ala Lys Arg Arg Xaa Ile Phe 1 5 83 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 83 His Ala Lys Arg Arg Ile Ile Phe 1 5 84 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 84 His Ala Lys Arg Arg Val Ile Phe 1 5 85 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 85 His Ala Lys Arg Arg Xaa Ile Phe 1 5 86 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 86 His Ala Lys Arg Arg Xaa Ile Phe 1 5 87 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 87 His Ala Lys Arg Arg Xaa Ile Phe 1 5 88 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 88 His Ala Lys Arg Arg Phe Ile Phe 1 5 89 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 89 His Ala Lys Arg Arg Xaa Ile Phe 1 5 90 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 90 His Ala Lys Arg Arg Leu Ala Phe 1 5 91 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 91 His Ala Lys Arg Arg Leu Leu Phe 1 5 92 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 92 His Ala Lys Arg Arg Leu Val Phe 1 5 93 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 93 His Ala Lys Arg Arg Leu Xaa Phe 1 5 94 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 94 His Ala Lys Arg Arg Leu Xaa Phe 1 5 95 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 95 His Ala Lys Arg Arg Leu Xaa Phe 1 5 96 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 96 His Ala Lys Arg Arg Leu Phe Phe 1 5 97 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 97 His Ala Lys Arg Arg Leu Xaa Phe 1 5 98 7 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 98 His Ala Lys Arg Arg Leu Phe 1 5 99 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 99 His Ala Lys Arg Arg Leu Ile Leu 1 5 100 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 100 His Ala Lys Arg Arg Leu Ile Xaa 1 5 101 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 101 His Ala Lys Arg Arg Leu Ile Xaa 1 5 102 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 102 His Ala Lys Arg Arg Leu Ile Tyr 1 5 103 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 103 His Ala Lys Arg Arg Leu Ile Xaa 1 5 104 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 104 His Ala Lys Arg Arg Leu Ile Xaa 1 5 105 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 105 His Ala Lys Arg Arg Leu Ile Trp 1 5 106 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 106 His Ala Lys Arg Arg Leu Ile Xaa 1 5 107 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 107 His Ala Lys Arg Arg Leu Ile Xaa 1 5 108 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 108 His Ala Lys Arg Arg Leu Ile Lys 1 5 109 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 109 His Ala Lys Arg Arg Leu Ile Xaa 1 5 110 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 110 His Ala Lys Arg Arg Leu Ile Xaa 1 5 111 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 111 His Ala Lys Arg Arg Leu Ile Xaa 1 5 112 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 112 His Ser Lys Arg Arg Leu Ile Xaa 1 5 113 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 113 His Ser Lys Arg Arg Leu Ile Xaa 1 5 114 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 114 His Ala Lys Arg Arg Leu Ile Xaa 1 5 115 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 115 His Ala Lys Arg Arg Leu Ile Xaa 1 5 116 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 116 His Ser Lys Arg Arg Leu Ile Xaa 1 5 117 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 117 His Ser Lys Arg Arg Leu Ile Xaa 1 5 118 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 118 His Ala Lys Arg Arg Leu Ile Xaa 1 5 119 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 119 His Ser Lys Arg Arg Leu Ile Xaa 1 5 120 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 120 His Ala Lys Arg Arg Leu Ile Xaa 1 5 121 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 121 His Ser Lys Arg Arg Leu Ile Xaa 1 5 122 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 122 Ala Ala Xaa Arg Arg Leu Ile Xaa 1 5 123 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 123 Ala Ala Lys Arg Arg Leu Ile Xaa 1 5 124 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 124 Ala Ala Lys Arg Xaa Leu Ile Xaa 1 5 125 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 125 Ala Ala Lys Arg Arg Leu Ala Xaa 1 5 126 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 126 Ala Ala Xaa Arg Ser Leu Ile Xaa 1 5 127 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 127 Ala Ala Lys Gln Arg Leu Ile Xaa 1 5 128 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 128 Ala Ala Lys Arg Arg Leu Ile Xaa 1 5 129 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 129 Gly Ala Lys Arg Arg Leu Ile Xaa 1 5 130 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 130 Ala Ala Lys Xaa Arg Leu Ile Xaa 1 5 131 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 131 Ala Ala Lys Ser Arg Leu Ile Xaa 1 5 132 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 132 Ala Ala Lys Xaa Arg Leu Ile Xaa 1 5 133 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 133 Ala Ala Lys Arg Lys Leu Ile Xaa 1 5 134 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 134 Ala Ala Lys Arg Xaa Leu Ile Xaa 1 5 135 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 135 Ala Ala Lys Arg Gln Leu Ile Xaa 1 5 136 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 136 Ala Ala Lys Arg Xaa Leu Ile Xaa 1 5 137 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 137 Ala Ala Lys Arg Thr Leu Ile Xaa 1 5 138 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 138 Ala Ala Lys Arg Xaa Leu Ile Xaa 1 5 139 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 139 Ala Ala Lys Arg Arg Xaa Ile Xaa 1 5 140 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 140 Ala Ala Lys Arg Arg Met Ile Xaa 1 5 141 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 141 Ala Ala Lys Arg Arg Ala Ile Xaa 1 5 142 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 142 Ala Ala Lys Arg Arg Xaa Ile Xaa 1 5 143 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 143 Ala Ala Lys Arg Arg Xaa Ile Xaa 1 5 144 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 144 Ala Ala Lys Arg Arg Xaa Ile Xaa 1 5 145 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 145 Ala Ala Lys Arg Arg Leu Gly Xaa 1 5 146 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 146 Ala Ala Lys Arg Arg Leu Xaa Xaa 1 5 147 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 147 Ala Ala Lys Arg Arg Leu Xaa Xaa 1 5 148 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 148 Ala Ala Lys Arg Arg Leu Xaa Xaa 1 5 149 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 149 Ala Ala Lys Arg Arg Leu Xaa Xaa 1 5 150 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 150 Ala Ala Lys Arg Arg Leu Pro Xaa 1 5 151 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 151 Ala Ala Lys Arg Arg Leu Xaa Xaa 1 5 152 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 152 Ala Ala Lys Arg Arg Leu Ser Xaa 1 5 153 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 153 Ala Ala Lys Arg Arg Leu Asp Xaa 1 5 154 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 154 Ala Ala Lys Arg Arg Leu Asn Xaa 1 5 155 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 155 Ala Ala Lys Arg Arg Leu Xaa Phe 1 5 156 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 156 Ala Ala Lys Arg Arg Leu Xaa Phe 1 5 157 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 157 Ala Ala Lys Arg Arg Leu Xaa Phe 1 5 158 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 158 Ala Ala Lys Arg Arg Leu Xaa Phe 1 5 159 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 159 Ala Ala Lys Arg Arg Leu Xaa Phe 1 5 160 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 160 Ala Ala Lys Arg Arg Leu Xaa Phe 1 5 161 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 161 Ala Ala Lys Arg Arg Leu Xaa Phe 1 5 162 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 162 Ala Ala Lys Arg Arg Leu Xaa Phe 1 5 163 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 163 Ala Ala Lys Arg Arg Leu Xaa Phe 1 5 164 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 164 Ala Ala Lys Arg Arg Leu Ile Xaa 1 5 165 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 165 Ala Ala Lys Arg Arg Leu Ile Xaa 1 5 166 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 166 Ala Ala Lys Arg Arg Leu Ile Xaa 1 5 167 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 167 Ala Ala Lys Arg Arg Leu Ile Xaa 1 5 168 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 168 Ala Ala Lys Arg Arg Leu Ile Xaa 1 5 169 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 169 Ala Ala Lys Arg Arg Leu Ile Xaa 1 5 170 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 170 Ala Ala Lys Arg Arg Leu Ile Xaa 1 5 171 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 171 Ala Ala Lys Arg Arg Leu Ile Xaa 1 5 172 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 172 Ala Ala Lys Arg Arg Leu Ile Xaa 1 5 173 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 173 His Ala Lys Arg Lys Leu Phe Gly 1 5 174 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 174 His Ala Lys Arg Xaa Leu Phe Gly 1 5 175 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 175 His Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 176 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 176 His Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 177 7 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 177 Ala Lys Arg Arg Leu Ile Xaa 178 8 PRT Artificial Sequence Description of Artificial Sequence p27 derived peptide 178 Ser Ala Cys Arg Asn Leu Phe Gly 1 5 179 8 PRT Artificial Sequence Description of Artificial Sequence Modelled cyclic peptide 179 Ser Ala Cys Arg Lys Leu Phe Gly 1 5 180 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 180 Ser Ala Cys Arg Asn Leu Phe Gly 1 5 181 7 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 181 Xaa Lys Arg Arg Leu Xaa Phe 1 5 182 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 182 His Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 183 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 183 Xaa Ala Lys Arg Arg Leu Ile Phe 1 5 184 4 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 184 Arg Leu Xaa Phe 1 185 4 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 185 Arg Leu Xaa Phe 1 186 9 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 186 Xaa His Ala Lys Arg Arg Leu Ile Phe 1 5 187 20 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 187 Thr Ser Met Thr Asp Phe Tyr His Ser Lys Arg Arg Leu Ile Phe Ser 1 5 10 15 Lys Arg Lys Pro 20 188 12 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 188 Thr Ser Met Thr Asp Phe Tyr His Ser Lys Arg Arg 1 5 10 189 12 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 189 Ser Met Thr Asp Phe Tyr His Ser Lys Arg Arg Leu 1 5 10 190 12 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 190 Met Thr Asp Phe Tyr His Ser Lys Arg Arg Leu Ile 1 5 10 191 12 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 191 Thr Asp Phe Tyr His Ser Lys Arg Arg Leu Ile Phe 1 5 10 192 12 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 192 Asp Phe Tyr His Ser Lys Arg Arg Leu Ile Phe Ser 1 5 10 193 12 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 193 Phe Tyr His Ser Lys Arg Arg Leu Ile Phe Ser Lys 1 5 10 194 12 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 194 Tyr His Ser Lys Arg Arg Leu Ile Phe Ser Lys Arg 1 5 10 195 12 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 195 His Ser Lys Arg Arg Leu Ile Phe Ser Lys Arg Lys 1 5 10 196 12 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 196 Ser Lys Arg Arg Leu Ile Phe Ser Lys Arg Lys Pro 1 5 10 197 12 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 197 Ala Phe Tyr His Ser Lys Arg Arg Leu Ile Phe Ser 1 5 10 198 12 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 198 Asp Ala Tyr His Ser Lys Arg Arg Leu Ile Phe Ser 1 5 10 199 12 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 199 Asp Phe Ala His Ser Lys Arg Arg Leu Ile Phe Ser 1 5 10 200 12 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 200 Asp Phe Tyr Ala Ser Lys Arg Arg Leu Ile Phe Ser 1 5 10 201 12 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 201 Asp Phe Tyr His Ala Lys Arg Arg Leu Ile Phe Ser 1 5 10 202 12 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 202 Asp Phe Tyr His Ser Ala Arg Arg Leu Ile Phe Ser 1 5 10 203 12 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 203 Asp Phe Tyr His Ser Lys Ala Arg Leu Ile Phe Ser 1 5 10 204 12 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 204 Asp Phe Tyr His Ser Lys Arg Ala Leu Ile Phe Ser 1 5 10 205 12 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 205 Asp Phe Tyr His Ser Lys Arg Arg Ala Ile Phe Ser 1 5 10 206 12 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 206 Asp Phe Tyr His Ser Lys Arg Arg Leu Ala Phe Ser 1 5 10 207 12 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 207 Asp Phe Tyr His Ser Lys Arg Arg Leu Ile Ala Ser 1 5 10 208 12 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 208 Asp Phe Tyr His Ser Lys Arg Arg Leu Ile Phe Ala 1 5 10 209 11 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 209 Phe Tyr His Ser Lys Arg Arg Leu Ile Phe Ser 1 5 10 210 10 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 210 Tyr His Ser Lys Arg Arg Leu Ile Phe Ser 1 5 10 211 9 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 211 His Ser Lys Arg Arg Leu Ile Phe Ser 1 5 212 11 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 212 Asp Phe Tyr Ala Ser Lys Arg Arg Leu Ile Phe 1 5 10 213 10 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 213 Asp Phe Tyr His Ser Lys Arg Arg Leu Ile 1 5 10 214 9 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 214 Asp Phe Tyr His Ser Lys Arg Arg Leu 1 5 215 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 215 Asp Phe Tyr His Ser Lys Arg Arg 1 5 216 7 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 216 Asp Phe Tyr His Ser Lys Arg 1 5 217 6 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 217 Asp Phe Tyr His Ser Lys 1 5 218 10 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 218 Phe Tyr His Ser Lys Arg Arg Leu Ile Phe 1 5 10 219 9 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 219 Phe Tyr His Ser Lys Arg Arg Leu Ile 1 5 220 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 220 Phe Tyr His Ser Lys Arg Arg Leu 1 5 221 7 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 221 Phe Tyr His Ser Lys Arg Arg 1 5 222 6 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 222 Phe Tyr His Ser Lys Arg 1 5 223 9 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 223 Tyr His Ser Lys Arg Arg Leu Ile Phe 1 5 224 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 224 Tyr His Ser Lys Arg Arg Leu Ile 1 5 225 7 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 225 Tyr His Ser Lys Arg Arg Leu 1 5 226 6 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 226 Tyr His Ser Lys Arg Arg 1 5 227 5 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 227 Tyr His Ser Lys Arg 1 5 228 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 228 His Ser Lys Arg Arg Leu Ile Phe 1 5 229 7 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 229 Ser Lys Arg Arg Leu Ile Phe 1 5 230 7 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 230 His Ser Lys Arg Arg Leu Ile 1 5 231 6 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 231 His Ser Lys Arg Arg Leu 1 5 232 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 232 Lys Arg Arg Leu Ile Phe Ser Lys 1 5 233 4 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 233 Xaa Xaa Arg Leu 1 234 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 234 Lys Ala Xaa Arg Arg Leu Ile Phe 1 5 235 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 235 His Ser Lys Arg Arg Leu Phe Gly 1 5 236 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 236 His Ser Lys Arg Arg Leu Asp Leu 1 5 237 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 237 His Ala Lys Arg Arg Leu Phe Gly 1 5 238 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 238 Pro Val Lys Arg Arg Leu Asp Leu 1 5 239 8 PRT Artificial Sequence Description of Artificial Sequence p107 peptide 239 Ser Ala Lys Arg Arg Leu Phe Gly 1 5 240 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 240 Lys Ala Xaa Arg Arg Leu Phe Gly 1 5 241 12 PRT Artificial Sequence SITE (1) Xaa = e-Aminohexanoic acid 241 Xaa Asp Phe Tyr His Ser Lys Arg Arg Leu Ile Phe 1 5 10 242 11 PRT Artificial Sequence Description of Artificial Sequence pRb-derived peptide 242 Asp Phe Tyr His Ala Lys Arg Arg Leu Ile Phe 1 5 10 243 15 PRT Artificial Sequence Description of Artificial Sequence pRb-derived peptide 243 Ser Asn Pro Pro Lys Pro Leu Lys Lys Leu Arg Phe Asp Ile Glu 1 5 10 15 244 8 PRT Artificial Sequence Description of Artificial Sequence pRb-derived peptide 244 Lys Pro Leu Lys Lys Leu Arg Phe 1 5 245 9 PRT Artificial Sequence SITE (1) Xaa = e-Aminohexanoic acid 245 Xaa Lys Pro Leu Lys Lys Leu Arg Phe 1 5 246 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 246 His Xaa Lys Arg Arg Leu Ile Phe 1 5 247 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 247 His Ala Xaa Arg Arg Leu Ile Phe 1 5 248 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 248 His Ala Lys Xaa Arg Leu Ile Phe 1 5 249 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 249 His Ala Lys Arg Xaa Leu Ile Phe 1 5 250 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 250 His Ala Lys Arg Arg Leu Xaa Phe 1 5 251 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 251 His Ala Lys Arg Arg Leu Ile Xaa 1 5 252 7 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 252 Xaa Xaa Xaa Xaa Xaa Leu Ile 1 5 253 7 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 253 Ala Lys Arg Arg Leu Ile Xaa 1 5 254 8 PRT Artificial Sequence Description of Artificial Sequence p21 derived peptide 254 Xaa Ala Xaa Xaa Xaa Leu Phe Gly 1 5 255 8 PRT Artificial Sequence Description of Artificial Sequence Peptide analogue 255 Ala Ala Lys Arg Arg Leu Phe Gly 1 5 256 8 PRT Artificial Sequence Description of Artificial Sequence Peptide analogue 256 Ala Ala Lys Xaa Arg Leu Phe Gly 1 5 257 8 PRT Artificial Sequence Description of Artificial Sequence Peptide analogue 257 Ala Ala Lys Ser Arg Leu Phe Gly 1 5 258 8 PRT Artificial Sequence Description of Artificial Sequence Peptide analogue 258 Ala Ala Lys Xaa Arg Leu Phe Gly 1 5 259 8 PRT Artificial Sequence Description of Artificial Sequence Peptide analogue 259 Ala Ala Lys Arg Lys Leu Phe Gly 1 5 260 8 PRT Artificial Sequence Description of Artificial Sequence Peptide analogue 260 Ala Ala Lys Arg Xaa Leu Phe Gly 1 5 261 8 PRT Artificial Sequence Description of Artificial Sequence Peptide analogue 261 Ala Ala Lys Arg Gln Leu Phe Gly 1 5 262 8 PRT Artificial Sequence Description of Artificial Sequence Peptide analogue 262 Ala Ala Lys Arg Xaa Leu Phe Gly 1 5 263 8 PRT Artificial Sequence Description of Artificial Sequence Peptide analogue 263 Ala Ala Lys Arg Thr Leu Phe Gly 1 5 264 8 PRT Artificial Sequence Description of Artificial Sequence Peptide analogue 264 Ala Ala Lys Arg Xaa Leu Phe Gly 1 5 265 8 PRT Artificial Sequence Description of Artificial Sequence Peptide analogue 265 Ala Ala Lys Arg Arg Met Phe Gly 1 5 266 8 PRT Artificial Sequence Description of Artificial Sequence Peptide analogue 266 Ala Ala Lys Arg Arg Ala Phe Gly 1 5 267 8 PRT Artificial Sequence Description of Artificial Sequence Peptide analogue 267 Ala Ala Lys Arg Arg Xaa Phe Gly 1 5 268 8 PRT Artificial Sequence Description of Artificial Sequence Peptide analogue 268 Ala Ala Lys Arg Arg Xaa Phe Gly 1 5 269 8 PRT Artificial Sequence Description of Artificial Sequence Peptide analogue 269 Ala Ala Lys Arg Arg Xaa Phe Gly 1 5 270 8 PRT Artificial Sequence Description of Artificial Sequence Peptide analogue 270 Ala Ala Lys Arg Arg Leu Xaa Gly 1 5 271 8 PRT Artificial Sequence Description of Artificial Sequence Peptide analogue 271 Ala Ala Lys Arg Arg Leu Xaa Gly 1 5 272 8 PRT Artificial Sequence Description of Artificial Sequence Peptide analogue 272 Ala Ala Lys Arg Arg Leu Xaa Gly 1 5 273 8 PRT Artificial Sequence Description of Artificial Sequence Peptide analogue 273 Ala Ala Lys Arg Arg Leu Xaa Gly 1 5 274 8 PRT Artificial Sequence Description of Artificial Sequence Peptide analogue 274 Ala Ala Lys Arg Arg Leu Xaa Gly 1 5 275 8 PRT Artificial Sequence Description of Artificial Sequence Peptide analogue 275 Ala Ala Lys Arg Arg Leu Xaa Gly 1 5 276 8 PRT Artificial Sequence Description of Artificial Sequence Peptide analogue 276 Ala Ala Lys Arg Arg Leu Xaa Gly 1 5 277 8 PRT Artificial Sequence Description of Artificial Sequence Peptide analogue 277 Ala Ala Lys Arg Arg Leu Xaa Gly 1 5 278 8 PRT Artificial Sequence Description of Artificial Sequence Peptide analogue 278 Ala Ala Lys Arg Arg Leu Xaa Gly 1 5 279 8 PRT Artificial Sequence Description of Artificial Sequence Peptide analogue 279 Ala Ala Lys Arg Arg Leu Xaa Gly 1 5 280 8 PRT Artificial Sequence Description of Artificial Sequence Peptide analogue 280 Ala Ala Lys Arg Arg Leu Xaa Gly 1 5 281 8 PRT Artificial Sequence Description of Artificial Sequence Peptide analogue 281 Ala Ala Lys Arg Arg Leu Xaa Gly 1 5 282 8 PRT Artificial Sequence Description of Artificial Sequence Peptide analogue 282 Ala Ala Lys Arg Arg Leu Xaa Gly 1 5 283 8 PRT Artificial Sequence Description of Artificial Sequence Peptide analogue 283 Ala Ala Lys Arg Arg Leu Phe Asp 1 5 284 8 PRT Artificial Sequence Description of Artificial Sequence Peptide analogue 284 Ala Ala Lys Arg Arg Leu Phe Glu 1 5 285 8 PRT Artificial Sequence Description of Artificial Sequence Peptide analogue 285 Ala Ala Lys Arg Arg Leu Phe Ser 1 5 286 8 PRT Artificial Sequence Description of Artificial Sequence Peptide analogue 286 Ala Ala Lys Arg Arg Leu Phe Asn 1 5 287 8 PRT Artificial Sequence Description of Artificial Sequence Peptide analogue 287 Ala Ala Lys Arg Arg Leu Phe Gln 1 5 288 8 PRT Artificial Sequence Description of Artificial Sequence Peptide analogue 288 Ala Ala Lys Arg Arg Leu Phe Lys 1 5 289 4 PRT Artificial Sequence Description of Artificial Sequence p27 derived peptide 289 Asn Leu Phe Gly 1 290 8 PRT Artificial Sequence p21 derived peptide 290 Ala Ala Xaa Arg Ser Leu Ile Gly 1 5 291 8 PRT Artificial Sequence p21 derived peptide 291 Ala Ala Xaa Arg Ser Leu Xaa Xaa 1 5 292 8 PRT Artificial Sequence p21 derived peptide 292 Ala Ala Xaa Arg Ser Leu Xaa Gly 1 5 293 5 PRT Artificial Sequence p21 derived peptide 293 Arg Xaa Xaa Xaa Xaa 1 5 294 5 PRT Artificial Sequence p21 derived peptide 294 Arg Arg Leu Asn Phe 1 5 295 5 PRT Artificial Sequence p21 derived peptide 295 Arg Arg Leu Asn Xaa 1 5 296 5 PRT Artificial Sequence p21 derived peptide 296 Arg Arg Leu Asn Xaa 1 5 297 5 PRT Artificial Sequence p21 derived peptide 297 Arg Arg Leu Ala Phe 1 5 298 5 PRT Artificial Sequence p21 derived peptide 298 Arg Arg Leu Ala Xaa 1 5 299 5 PRT Artificial Sequence p21 derived peptide 299 Arg Arg Leu Ala Xaa 1 5 300 5 PRT Artificial Sequence p21 derived peptide 300 Arg Arg Leu Gly Phe 1 5 301 5 PRT Artificial Sequence p21 derived peptide 301 Arg Arg Leu Gly Xaa 1 5 302 5 PRT Artificial Sequence p21 derived peptide 302 Arg Arg Leu Gly Xaa 1 5 303 5 PRT Artificial Sequence p21 derived peptide 303 Arg Arg Ile Asn Phe 1 5 304 5 PRT Artificial Sequence p21 derived peptide 304 Arg Arg Ile Asn Xaa 1 5 305 5 PRT Artificial Sequence p21 derived peptide 305 Arg Arg Ile Asn Xaa 1 5 306 5 PRT Artificial Sequence p21 derived peptide 306 Arg Arg Ile Ala Phe 1 5 307 5 PRT Artificial Sequence p21 derived peptide 307 Arg Arg Ile Ala Xaa 1 5 308 5 PRT Artificial Sequence p21 derived peptide 308 Arg Arg Ile Ala Xaa 1 5 309 5 PRT Artificial Sequence p21 derived peptide 309 Arg Arg Ile Gly Phe 1 5 310 5 PRT Artificial Sequence p21 derived peptide 310 Arg Arg Ile Gly Xaa 1 5 311 5 PRT Artificial Sequence p21 derived peptide 311 Arg Arg Ile Gly Xaa 1 5 312 5 PRT Artificial Sequence p21 derived peptide 312 Arg Arg Val Asn Phe 1 5 313 5 PRT Artificial Sequence p21 derived peptide 313 Arg Arg Val Asn Xaa 1 5 314 5 PRT Artificial Sequence p21 derived peptide 314 Arg Arg Val Asn Xaa 1 5 315 5 PRT Artificial Sequence p21 derived peptide 315 Arg Arg Val Ala Phe 1 5 316 5 PRT Artificial Sequence p21 derived peptide 316 Arg Arg Val Ala Xaa 1 5 317 5 PRT Artificial Sequence p21 derived peptide 317 Arg Arg Val Ala Xaa 1 5 318 5 PRT Artificial Sequence p21 derived peptide 318 Arg Arg Val Gly Phe 1 5 319 5 PRT Artificial Sequence p21 derived peptide 319 Arg Arg Val Gly Xaa 1 5 320 5 PRT Artificial Sequence p21 derived peptide 320 Arg Arg Val Gly Xaa 1 5 321 5 PRT Artificial Sequence p21 derived peptide 321 Arg Ser Leu Asn Phe 1 5 322 5 PRT Artificial Sequence p21 derived peptide 322 Arg Ser Leu Asn Xaa 1 5 323 5 PRT Artificial Sequence p21 derived peptide 323 Arg Ser Leu Asn Xaa 1 5 324 5 PRT Artificial Sequence p21 derived peptide 324 Arg Ser Leu Ala Phe 1 5 325 5 PRT Artificial Sequence p21 derived peptide 325 Arg Ser Leu Ala Xaa 1 5 326 5 PRT Artificial Sequence p21 derived peptide 326 Arg Ser Leu Ala Xaa 1 5 327 5 PRT Artificial Sequence p21 derived peptide 327 Arg Ser Leu Gly Phe 1 5 328 5 PRT Artificial Sequence p21 derived peptide 328 Arg Ser Leu Gly Xaa 1 5 329 5 PRT Artificial Sequence p21 derived peptide 329 Arg Ser Leu Gly Xaa 1 5 330 5 PRT Artificial Sequence p21 derived peptide 330 Arg Ser Ile Asn Phe 1 5 331 5 PRT Artificial Sequence p21 derived peptide 331 Arg Ser Ile Asn Xaa 1 5 332 5 PRT Artificial Sequence p21 derived peptide 332 Arg Ser Ile Asn Xaa 1 5 333 5 PRT Artificial Sequence p21 derived peptide 333 Arg Ser Ile Ala Phe 1 5 334 5 PRT Artificial Sequence p21 derived peptide 334 Arg Ser Ile Ala Xaa 1 5 335 5 PRT Artificial Sequence p21 derived peptide 335 Arg Ser Ile Ala Xaa 1 5 336 5 PRT Artificial Sequence p21 derived peptide 336 Arg Ser Ile Gly Phe 1 5 337 5 PRT Artificial Sequence p21 derived peptide 337 Arg Ser Ile Gly Xaa 1 5 338 5 PRT Artificial Sequence p21 derived peptide 338 Arg Ser Ile Gly Xaa 1 5 339 5 PRT Artificial Sequence p21 derived peptide 339 Arg Ser Val Asn Phe 1 5 340 5 PRT Artificial Sequence p21 derived peptide 340 Arg Ser Val Asn Xaa 1 5 341 5 PRT Artificial Sequence p21 derived peptide 341 Arg Ser Val Asn Xaa 1 5 342 5 PRT Artificial Sequence p21 derived peptide 342 Arg Ser Val Ala Phe 1 5 343 5 PRT Artificial Sequence p21 derived peptide 343 Arg Ser Val Ala Xaa 1 5 344 5 PRT Artificial Sequence p21 derived peptide 344 Arg Ser Val Ala Xaa 1 5 345 5 PRT Artificial Sequence p21 derived peptide 345 Arg Ser Val Gly Phe 1 5 346 5 PRT Artificial Sequence p21 derived peptide 346 Arg Ser Val Gly Xaa 1 5 347 5 PRT Artificial Sequence p21 derived peptide 347 Arg Ser Val Gly Xaa 1 5 348 5 PRT Artificial Sequence p21 derived peptide 348 Arg Lys Leu Asn Phe 1 5 349 5 PRT Artificial Sequence p21 derived peptide 349 Arg Lys Leu Asn Xaa 1 5 350 5 PRT Artificial Sequence p21 derived peptide 350 Arg Lys Leu Asn Xaa 1 5 351 5 PRT Artificial Sequence p21 derived peptide 351 Arg Lys Leu Ala Phe 1 5 352 5 PRT Artificial Sequence p21 derived peptide 352 Arg Lys Leu Ala Xaa 1 5 353 5 PRT Artificial Sequence p21 derived peptide 353 Arg Lys Leu Ala Xaa 1 5 354 5 PRT Artificial Sequence p21 derived peptide 354 Arg Lys Leu Gly Phe 1 5 355 5 PRT Artificial Sequence p21 derived peptide 355 Arg Lys Leu Gly Xaa 1 5 356 5 PRT Artificial Sequence p21 derived peptide 356 Arg Lys Leu Gly Xaa 1 5 357 5 PRT Artificial Sequence p21 derived peptide 357 Arg Lys Ile Asn Phe 1 5 358 5 PRT Artificial Sequence p21 derived peptide 358 Arg Lys Ile Asn Xaa 1 5 359 5 PRT Artificial Sequence p21 derived peptide 359 Arg Lys Ile Asn Xaa 1 5 360 5 PRT Artificial Sequence p21 derived peptide 360 Arg Lys Ile Ala Phe 1 5 361 5 PRT Artificial Sequence p21 derived peptide 361 Arg Lys Ile Ala Xaa 1 5 362 5 PRT Artificial Sequence p21 derived peptide 362 Arg Lys Ile Ala Xaa 1 5 363 5 PRT Artificial Sequence p21 derived peptide 363 Arg Lys Ile Gly Phe 1 5 364 5 PRT Artificial Sequence p21 derived peptide 364 Arg Lys Ile Gly Xaa 1 5 365 5 PRT Artificial Sequence p21 derived peptide 365 Arg Lys Ile Gly Xaa 1 5 366 5 PRT Artificial Sequence p21 derived peptide 366 Arg Lys Val Asn Phe 1 5 367 5 PRT Artificial Sequence p21 derived peptide 367 Arg Lys Val Asn Xaa 1 5 368 5 PRT Artificial Sequence p21 derived peptide 368 Arg Lys Val Asn Xaa 1 5 369 5 PRT Artificial Sequence p21 derived peptide 369 Arg Lys Val Ala Phe 1 5 370 5 PRT Artificial Sequence p21 derived peptide 370 Arg Lys Val Ala Xaa 1 5 371 5 PRT Artificial Sequence p21 derived peptide 371 Arg Lys Val Ala Xaa 1 5 372 5 PRT Artificial Sequence p21 derived peptide 372 Arg Lys Val Gly Phe 1 5 373 5 PRT Artificial Sequence p21 derived peptide 373 Arg Lys Val Gly Xaa 1 5 374 5 PRT Artificial Sequence p21 derived peptide 374 Arg Lys Val Gly Xaa 1 5 375 5 PRT Artificial Sequence p21 derived peptide 375 Arg Arg Leu Ile Xaa 1 5 376 5 PRT Artificial Sequence p21 derived peptide 376 Xaa Xaa Leu Ile Xaa 1 5 377 5 PRT Artificial Sequence p21 derived peptide 377 Arg Arg Leu Ile Phe 1 5 378 5 PRT Artificial Sequence p21 derived peptide 378 Arg Arg Leu Asn Phe 1 5 379 5 PRT Artificial Sequence p21 derived peptide 379 Arg Arg Leu Asn Xaa 1 5 380 5 PRT Artificial Sequence p21 derived peptide 380 Arg Arg Leu Asn Xaa 1 5 381 5 PRT Artificial Sequence p21 derived peptide 381 Arg Arg Leu Ala Phe 1 5 382 5 PRT Artificial Sequence p21 derived peptide 382 Arg Arg Leu Ala Xaa 1 5 383 5 PRT Artificial Sequence p21 derived peptide 383 Arg Arg Leu Ala Xaa 1 5 384 5 PRT Artificial Sequence p21 derived peptide 384 Arg Arg Leu Gly Phe 1 5 385 5 PRT Artificial Sequence p21 derived peptide 385 Arg Arg Leu Gly Xaa 1 5 386 5 PRT Artificial Sequence p21 derived peptide 386 Arg Arg Leu Gly Xaa 1 5 387 5 PRT Artificial Sequence p21 derived peptide 387 Arg Arg Ile Asn Phe 1 5 388 5 PRT Artificial Sequence p21 derived peptide 388 Arg Arg Ile Asn Xaa 1 5 389 5 PRT Artificial Sequence p21 derived peptide 389 Arg Arg Ile Asn Xaa 1 5 390 5 PRT Artificial Sequence p21 derived peptide 390 Arg Arg Ile Ala Phe 1 5 391 5 PRT Artificial Sequence p21 derived peptide 391 Arg Arg Ile Ala Xaa 1 5 392 5 PRT Artificial Sequence p21 derived peptide 392 Arg Arg Ile Ala Xaa 1 5 393 5 PRT Artificial Sequence p21 derived peptide 393 Arg Arg Ile Gly Phe 1 5 394 5 PRT Artificial Sequence p21 derived peptide 394 Arg Arg Ile Gly Xaa 1 5 395 5 PRT Artificial Sequence p21 derived peptide 395 Arg Arg Ile Gly Xaa 1 5 396 5 PRT Artificial Sequence p21 derived peptide 396 Arg Arg Val Asn Phe 1 5 397 5 PRT Artificial Sequence p21 derived peptide 397 Arg Arg Val Asn Xaa 1 5 398 5 PRT Artificial Sequence p21 derived peptide 398 Arg Arg Val Asn Xaa 1 5 399 5 PRT Artificial Sequence p21 derived peptide 399 Arg Arg Val Ala Phe 1 5 400 5 PRT Artificial Sequence p21 derived peptide 400 Arg Arg Val Ala Xaa 1 5 401 5 PRT Artificial Sequence p21 derived peptide 401 Arg Arg Val Ala Xaa 1 5 402 5 PRT Artificial Sequence p21 derived peptide 402 Arg Arg Val Gly Phe 1 5 403 5 PRT Artificial Sequence p21 derived peptide 403 Arg Arg Val Gly Xaa 1 5 404 5 PRT Artificial Sequence p21 derived peptide 404 Arg Arg Val Gly Xaa 1 5 405 5 PRT Artificial Sequence p21 derived peptide 405 Arg Ser Leu Asn Phe 1 5 406 5 PRT Artificial Sequence p21 derived peptide 406 Arg Ser Leu Asn Xaa 1 5 407 5 PRT Artificial Sequence p21 derived peptide 407 Arg Ser Leu Asn Xaa 1 5 408 5 PRT Artificial Sequence p21 derived peptide 408 Arg Ser Leu Ala Phe 1 5 409 5 PRT Artificial Sequence p21 derived peptide 409 Arg Ser Leu Ala Xaa 1 5 410 5 PRT Artificial Sequence p21 derived peptide 410 Arg Ser Leu Ala Xaa 1 5 411 5 PRT Artificial Sequence p21 derived peptide 411 Arg Ser Leu Gly Phe 1 5 412 5 PRT Artificial Sequence p21 derived peptide 412 Arg Ser Leu Gly Xaa 1 5 413 5 PRT Artificial Sequence p21 derived peptide 413 Arg Ser Leu Gly Xaa 1 5 414 5 PRT Artificial Sequence p21 derived peptide 414 Arg Ser Ile Asn Phe 1 5 415 5 PRT Artificial Sequence p21 derived peptide 415 Arg Ser Ile Asn Xaa 1 5 416 5 PRT Artificial Sequence p21 derived peptide 416 Arg Ser Ile Asn Xaa 1 5 417 5 PRT Artificial Sequence p21 derived peptide 417 Arg Ser Ile Ala Phe 1 5 418 5 PRT Artificial Sequence p21 derived peptide 418 Arg Ser Ile Ala Xaa 1 5 419 5 PRT Artificial Sequence p21 derived peptide 419 Arg Ser Ile Ala Xaa 1 5 420 5 PRT Artificial Sequence p21 derived peptide 420 Arg Ser Ile Gly Phe 1 5 421 5 PRT Artificial Sequence p21 derived peptide 421 Arg Ser Ile Gly Xaa 1 5 422 5 PRT Artificial Sequence p21 derived peptide 422 Arg Ser Ile Gly Xaa 1 5 423 5 PRT Artificial Sequence p21 derived peptide 423 Arg Ser Val Asn Phe 1 5 424 5 PRT Artificial Sequence p21 derived peptide 424 Arg Ser Val Asn Xaa 1 5 425 5 PRT Artificial Sequence p21 derived peptide 425 Arg Ser Val Asn Xaa 1 5 426 5 PRT Artificial Sequence p21 derived peptide 426 Arg Ser Val Ala Phe 1 5 427 5 PRT Artificial Sequence p21 derived peptide 427 Arg Ser Val Ala Xaa 1 5 428 5 PRT Artificial Sequence p21 derived peptide 428 Arg Ser Val Ala Xaa 1 5 429 5 PRT Artificial Sequence p21 derived peptide 429 Arg Ser Val Gly Phe 1 5 430 5 PRT Artificial Sequence p21 derived peptide 430 Arg Ser Val Gly Xaa 1 5 431 5 PRT Artificial Sequence p21 derived peptide 431 Arg Ser Val Gly Xaa 1 5 432 5 PRT Artificial Sequence p21 derived peptide 432 Arg Lys Leu Asn Phe 1 5 433 5 PRT Artificial Sequence p21 derived peptide 433 Arg Lys Leu Asn Xaa 1 5 434 5 PRT Artificial Sequence p21 derived peptide 434 Arg Lys Leu Asn Xaa 1 5 435 5 PRT Artificial Sequence p21 derived peptide 435 Arg Lys Leu Ala Phe 1 5 436 5 PRT Artificial Sequence p21 derived peptide 436 Arg Lys Leu Ala Xaa 1 5 437 5 PRT Artificial Sequence p21 derived peptide 437 Arg Lys Leu Ala Xaa 1 5 438 5 PRT Artificial Sequence p21 derived peptide 438 Arg Lys Leu Gly Phe 1 5 439 5 PRT Artificial Sequence p21 derived peptide 439 Arg Lys Leu Gly Xaa 1 5 440 5 PRT Artificial Sequence p21 derived peptide 440 Arg Lys Leu Gly Xaa 1 5 441 5 PRT Artificial Sequence p21 derived peptide 441 Arg Lys Ile Asn Phe 1 5 442 5 PRT Artificial Sequence p21 derived peptide 442 Arg Lys Ile Asn Xaa 1 5 443 5 PRT Artificial Sequence p21 derived peptide 443 Arg Lys Ile Asn Xaa 1 5 444 5 PRT Artificial Sequence p21 derived peptide 444 Arg Lys Ile Ala Phe 1 5 445 5 PRT Artificial Sequence p21 derived peptide 445 Arg Lys Ile Ala Xaa 1 5 446 5 PRT Artificial Sequence p21 derived peptide 446 Arg Lys Ile Ala Xaa 1 5 447 5 PRT Artificial Sequence p21 derived peptide 447 Arg Lys Ile Gly Phe 1 5 448 5 PRT Artificial Sequence p21 derived peptide 448 Arg Lys Ile Gly Xaa 1 5 449 5 PRT Artificial Sequence p21 derived peptide 449 Arg Lys Ile Gly Xaa 1 5 450 5 PRT Artificial Sequence p21 derived peptide 450 Arg Lys Val Asn Phe 1 5 451 5 PRT Artificial Sequence p21 derived peptide 451 Arg Lys Val Asn Xaa 1 5 452 5 PRT Artificial Sequence p21 derived peptide 452 Arg Lys Val Asn Xaa 1 5 453 5 PRT Artificial Sequence p21 derived peptide 453 Arg Lys Val Ala Phe 1 5 454 5 PRT Artificial Sequence p21 derived peptide 454 Arg Lys Val Ala Xaa 1 5 455 5 PRT Artificial Sequence p21 derived peptide 455 Arg Lys Val Ala Xaa 1 5 456 5 PRT Artificial Sequence p21 derived peptide 456 Arg Lys Val Gly Phe 1 5 457 5 PRT Artificial Sequence p21 derived peptide 457 Arg Lys Val Gly Xaa 1 5 458 5 PRT Artificial Sequence p21 derived peptide 458 Arg Lys Val Gly Xaa 1 5 459 5 PRT Artificial Sequence p21 derived peptide 459 Arg Arg Leu Ile Xaa 1 5 

What is claimed is:
 1. A peptide of formula I N₁DFYHSKRRLIFN₂  (formula I) (SEQ ID No. 4), comprising the motif XLXF, wherein N₁ and N₂ are independently a natural or non-natural amino acid or nothing; or the peptide of formula I having up to 8 amino acid residues deleted from the N-terminal end; and variants thereof wherein at least one amino acid residue is replaced by an alternative natural or non-natural replacement amino acid residue, with the proviso that the motif XLXF is retained, wherein X refers to any natural or unnatural amino acid.
 2. A peptide according to claim 1, wherein N₁ and N₂ are independently selected from nothing and the polar residues C, N, Q, S, T and Y.
 3. A peptide according to claim 2, wherein N₁ is a natural or unnatural amino acid.
 4. A peptide according to claim 3, wherein N₁ is threonine.
 5. A peptide according to claim 2, wherein N₂ is a natural or unnatural amino acid.
 6. A peptide according to claim 3, wherein N₁ is serine.
 7. A peptide according to claim 1, wherein up to 6 amino acid residues are deleted from the N-terminal end of the peptide of formula I.
 8. A peptide according to claim 7, wherein from 3-5 amino acid residues are deleted from the N-terminal end of the peptide of formula I.
 9. A peptide according to claim 8, wherein 4 amino acid residues are deleted from the N-terminal end of the peptide of formula I.
 10. A peptide according to claim 7, wherein N₂ is a natural or unnatural amino acid.
 11. A peptide according to claim 10, wherein N₂ is serine.
 12. A peptide according to claim 1, wherein 7 or 8 amino acid residues are deleted from the N-terminal end of the peptide of formula I.
 13. A peptide of formula DFYHSKRRLIF  (SEQ ID No. 1), comprising the motif XLXF, or such a peptide (i) bearing a further amino acid residue at either end; and, (ii) having up to 7 amino acid residues deleted from the N-terminal end; and variants thereof wherein at least one amino acid residue is replaced by an alternative natural or unnatural replacement amino acid residue, with the proviso that the motif XLXF is retained, wherein the peptide of SEQ ID No. 1 is modified by at least one of; deletion, addition or substitution of one or more amino acid residues, or by substitution of one or more natural amino acid residues by the corresponding D-stereomer or by a non-natural amino acid residue, chemical derivatives of the peptides, cyclic peptides derived from the peptides or from the peptide derivatives, dual peptides, multimers of the peptides and any of said peptides in the D-stereomer form, or the order of the final two residues at the C-terminal end are reversed.
 14. A variant according to claim 13, wherein the serine residue corresponding to p21(153Ser), is replaced by an alanine residue.
 15. A peptide according to claim 13, selected from; D F Y H S K R R L I F S (Seq. I. D. # 4) T D F Y H S K R R L I F, (Seq. I. D. # 5) A F Y H S K R R L I F S, (Seq. I. D. # 6) D A Y H S K R R L I F S, (Seq. I. D. # 7) D F A H S K R R L I F S, (Seq. I. D. # 8) D F Y A S K R R L I F S, (Seq. I. D. # 9) D F Y H A K R R L I F S, (Seq. I. D. # 10) D F Y H S A R R L I F S, (Seq. I. D. # 11) D F Y H S K R A L I F S, (Seq. I. D. # 12) D F Y H S K R R L A F S, (Seq. I. D. # 13) D F Y H S K R R L I F A, (Seq. I. D. # 14) F Y H S K R R L I F S, (Seq. I. D. # 15) Y H S K R R L I F S, (Seq. I. D. # 16) H S K R R L I F S, (Seq. I. D. # 17) D F Y H S K R R L I F, (Seq. I. D. # 18) F Y H S K R R L I F (Seq. I. D. # 19) Y H S K R R L I F (Seq. I. D. # 20) H S K R R L I F, (Seq. I. D. # 21) S K R R L I F, (Seq. I. D. # 22) K R R L I F, (Seq. I. D. # 23) H- Arg- Leu- Ile- Phe -NH2 (Seq. I. D. # 24) H- Arg- Arg- Leu- Ile- Phe -NH2 (Seq. I. D. # 25) H- Lys- Arg- Arg- Leu- Ile- Phe -NH2 (Seq. I. D. # 26) H- Ala- Lys- Arg- Arg- Leu- Ile- Phe -NH2 (Seq. I. D. # 27) H- His- Ala- Lys- Arg- Arg- Leu- Ile- Phe -NH2 (Seq. I. D. # 28) H- Asn- Leu- Phe- Gly -NH2 (Seq. I. D. # 29) H- Arg- Asn- Leu- Phe- Gly -NH2 (Seq. I. D. # 30) H- Abu- Arg- Asn- Leu- Phe- Gly -NH2 (Seq. I. D. # 31) H- Ala- Abu- Arg- Asn- Leu- Phe- Gly -NH2 and (Seq. I. D. # 32) H- Ser- Ala- Abu- Arg- Asn- Leu- Phe- Gly -NH2 (Seq. I. D. # 33)


16. A peptide of formula II; X₁X₂X₃RX₄LX₅F  (formula II) (SEQ ID No. 2) wherein X₁, X₃, X₄ and X₅ may be any amino acid and X₂ is serine or alanine; and variants thereof.
 17. A peptide according to claim 16, wherein X₅ is selected from isoleucine and glycine.
 18. A peptide according to claim 16, wherein X₁ and X₄ are both basic amino acid residues and X₃ is a basic or polar residue.
 19. A peptide according to claim 18, wherein X₁ is histidine and X₄ is arginine, and X₃ is lysine or cysteine.
 20. A peptide of formula; X₁X₂X₃RX₄LX₅F  (SEQ ID No. 2) wherein X₁, X₃, X₄ and X₅ may be any amino acid and X₂ is serine or alanine; and variants thereof, wherein the peptide is modified by at least one of a deletion, addition or substitution of one or more amino acid residues, or by substitution of one or more natural amino acid residues by the corresponding D-stereomer or by a non-natural amino acid residue, chemical derivatives of the peptides, cyclic peptides derived from the peptides or from the peptide derivatives, dual peptides, multimers of the peptides and any of said peptides in the D-stereomer form, or the order of the final two residues at the C-terminal end are reversed.
 21. A peptide of formula; X₁X₂X₃RX₄LX₅F  (SEQ ID No. 2) wherein X₁, X₃, X₄ and X₅ may be any amino acid and X₂ is serine or alanine; and variants thereof, wherein: (a) X₁ is deleted or is any amino acid, (b) X₂ is serine or alanine or a straight or branched chain amino acid, (c) X₃ is a basic amino acid or straight chain aliphatic amino acid, (d) R is unchanged or conservatively substituted (by basic amino acids), (e) X₄ is any amino acid that is capable of providing at least one site for participating in hydrogen bonding, (f) L is unchanged or conservatively substituted, (g) X₅ is any amino acid, or (h) F is unchanged or substituted by any aromatic amino acid.
 22. A peptide of formula; X₁X₂X₃RX₄LX₅F  (SEQ ID No. 2), wherein (a) X₁ is histidine, deleted or replaced by a natural or unnatural amino acid residue such as alanine, 3-pyridylalanine (Pya), 2-thienylalanine (Thi), homoserine (Hse), phenylalanine, or diaminobutyric acid (Dab), (b) X₂ is alanine or an alternative natural or unnatural amino acid residue having a smaller or slightly larger aromatic or aliphatic side chain, such as glycine, aminobutyric acid (Abu), norvaline (Nva), t-butylglycine(Bug), valine, isoleucine, phenylglycine (Phg) or phenylalanine, (c) X₃ is lysine or either a basic residue such as arginine or an uncharged natural or unnatural amino acid residue, such as norleucine (Nle), aminobutyric acid (Abu) or leucine, (d) arginine is replaced by either a basic residue such as lysine or an uncharged natural or unnatural amino acid residue, such as citrulline (Cit), homoserine, histidine, norleucine (Nle) or glutamine, (e) X₄ is arginine or a natural or unnatural amino acid residue, such as asparagine, proline, serine, aminoisobutyric acid (Aib) or sarcosine (Sar), or an amino acid residue capable of forming a cyclic linkage such as lysine or ornithine, (f) leucine is replaced with a natural or unnatural amino acid residue having a slightly larger aromatic or aliphatic side chain, such as norleucine, norvaline, cyclohexylalanine (Cha), phenylalanine or 1-naphthylalanine (1Nal), (g) X₅ is isoleucine or an alternative natural or unnatural amino acid residue having a slightly larger aromatic or aliphatic side chain, such as norleucine, norvaline, cyclohexylalanine (Cha), phenylalanine or 1-naphthylalanine (1Nal), (h) phenylalanine is replaced with a natural or unnatural amino acid such as leucine, cyclohexylalanine (Cha), homophenylalanine (Hof), tyrosine, para-fluorophenylalanine (pFPhe), meta-fluorophenylalanine (mFPhe), trptophan, 1-naphthylalanine (1Nal), 2-naphthylalanine (2Nal), biphenylalanine (Bip) or (Tic), (i) X₅ and the terminal phenylalanine residue are reversed, or (j) the peptide is in cyclic form by the formation of a linkage between the side chain of X₄ and the C-terminus residue.
 23. A peptide according to claim 16, wherein X₂ is alanine.
 24. A peptide according to claim 16, wherein X₅ is isoleuc Seq. I.D. # ine.
 25. A peptide according to claim 20, selected from the group consisting of: HSKRRLIF (Seq. I.D. # 34) HAKRRLIF (Seq. I.D. # 35) HSKRRLFG (Seq. I.D. # 36) HAKRRLFG (Seq. I.D. # 37) KACRRLFG (Seq. I.D. # 38) KACRRLIF (Seq. I.D. # 39) X1 X2 X3 R X4 L X5 F H- His- Ala- Lys- Arg- Arg- Leu- Ile- Phe -NH2 ? H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 40) H- Ala- Lys- Arg- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 41) H- Pya- Ala- Lys- Arg- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 42) H- Thi- Ala- Lys- Arg- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 43) H- Hse- Ala- Lys- Arg- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 44) H- Phe- Ala- Lys- Arg- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 45) H- Dab- Ala- Lys- Arg- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 46) H- His- Gly- Lys- Arg- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 47) H- His- Abu- Lys- Arg- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 48) H- His- Nva- Lys- Arg- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 49) H- His- Bug- Lys- Arg- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 50) H- His- Val- Lys- Arg- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 51) H- His- Ile- Lys- Arg- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 52) H- His- Phg- Lys- Arg- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 53) H- His- Phe- Lys- Arg- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 54) H- His- Ala- Ala- Arg- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 56) H- His- Ala- Nle- Arg- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 57) H- His- Ala- Abu- Arg- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 58) H- His- Ala- Leu- Arg- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 59) H- His- Ala- Arg- Arg- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 60) H- His- Ala- Lys- Ala- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 61) H- His- Ala- Lys- Cit- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 62) H- His- Ala- Lys- Hse- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 63) H- His- Ala- Lys- His- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 64) H- His- Ala- Lys- Nle- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 65) H- His- Ala- Lys- Gln- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 66) H- His- Ala- Lys- Lys- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 67) H- His- Ala- Lys- Arg- Ala- Leu- Ile- Phe -NH2 (Seq. I.D. # 68) H- His- Ala- Lys- Arg- Asn- Leu- Ile- Phe -NH2 (Seq. I.D. # 69) H- His- Ala- Lys- Arg- Pro- Leu- Ile- Phe -NH2 (Seq. I.D. # 70) H- His- Ala- Lys- Arg- Ser- Leu- Ile- Phe -NH2 (Seq. I.D. # 71) H- His- Ala- Lys- Arg- Aib- Leu- Ile- Phe -NH2 (Seq. I.D. # 72) H- His- Ala- Lys- Arg- Sar- Leu- Ile- Phe -NH2 (Seq. I.D. # 73) H- His- Ala- Lys- Arg- Cit- Leu- Ile- Phe -NH2 (Seq. I.D. # 74) H- His- Ala- Lys- Arg- Arg- Ala- Ile- Phe -NH2 (Seq. I.D. # ??) H- His- Ala- Lys- Arg- Arg- leu- Ile- Phe -NH2 (Seq. I.D. # 75) H- His- Ala- Lys- Arg- Arg- Ile- Ile- Phe -NH2 (Seq. I.D. # ??) H- His- Ala- Lys- Arg- Arg- Val- Ile- Phe -NH2 (Seq. I.D. # 79) H- His- Ala- Lys- Arg- Arg- Nle- Ile- Phe -NH2 (Seq. I.D. # 80) H- His- Ala- Lys- Arg- Arg- Nva- Ile- Phe -NH2 (Seq. I.D. # 81) H- His- Ala- Lys- Arg- Arg- Cha- Ile- Phe -NH2 (Seq. I.D. # 82) H- His- Ala- Lys- Arg- Arg- Phe- Ile- Phe -NH2 (Seq. I.D. # 83) H- His- Ala- Lys- Arg- Arg- 1Nap- Ile- Phe -NH2 (Seq. I.D. # 84) H- His- Ala- Lys- Arg- Arg- Leu- Ala- Phe -NH2 (Seq. I.D. # 85) H- His- Ala- Lys- Arg- Arg- Leu- Leu- Phe -NH2 (Seq. I.D. # 86) H- His- Ala- Lys- Arg- Arg- Leu- Val- Phe -NH2 (Seq. I.D. # 87) H- His- Ala- Lys- Arg- Arg- Leu- Nle- Phe -NH2 (Seq. I.D. # 88) H- His- Ala- Lys- Arg- Arg- Leu- Nva- Phe -NH2 (Seq. I.D. # 89) H- His- Ala- Lys- Arg- Arg- Leu- Cha- Phe -NH2 (Seq. I.D. # 90) H- His- Ala- Lys- Arg- Arg- Leu- Phe- Phe -NH2 (Seq. I.D. # 91) H- His- Ala- Lys- Arg- Arg- Leu- 1Nap- Phe -NH2 (Seq. I.D. # 92) H- His- Ala- Lys- Arg- Arg- Leu- Phe -NH2 (Seq. I.D. # 93) H- His- Ala- Lys- Arg- Arg- Leu- Ile- Leu -NH2 (Seq. I.D. # 95) H- His- Ala- Lys- Arg- Arg- Leu- Ile- Cha -NH2 (Seq. I.D. # 96) H- His- Ala- Lys- Arg- Arg- Leu- Ile- Hof -NH2 (Seq. I.D. # 97) H- His- Ala- Lys- Arg- Arg- Leu- Ile- Tyr -NH2 (Seq. I.D. # 98) H- His- Ala- Lys- Arg- Arg- Leu- Ile- pFPhe -NH2 (Seq. I.D. # 99) H- His- Ala- Lys- Arg- Arg- Leu- Ile- mFPhe -NH2 (Seq. I.D. # 100) H- His- Ala- Lys- Arg- Arg- Leu- Ile- Trp -NH2 (Seq. I.D. # 101) H- His- Ala- Lys- Arg- Arg- Leu- Ile- 1Nap -NH2 (Seq. I.D. # 102) H- His- Ala- Lys- Arg- Arg- Leu- Ile- 2Nap -NH2 (Seq. I.D. # 103) H- His- Ala- Lys- Arg- Arg- Leu- Ile- Lys -NH2 (Seq. I.D. # 104) H- His- Ala- Lys- Arg- Arg- Leu- Ile- Tic -NH2 (Seq. I.D. # 105) H- His Ala Lys Arg Arg Leu Ile L-Pse OH (Seq. I.D. # 106) H- His Ala Lys Arg Arg Leu Ile D-Pse OH (Seq. I.D. # 107) H- His Ser Lys Arg Arg Leu Ile L-Pse OH (Seq. I.D. # 108) H- His Ser Lys Arg Arg Leu Ile D-Pse OH (Seq. I.D. # 109) H- His Ala Lys Arg Arg Leu Ile L-Psa OH (Seq. I.D. # 110) H- His Ala Lys Arg Arg Leu Ile D-Psa OH (Seq. I.D. # 111) H- His Ser Lys Arg Arg Leu Ile L-Psa OH (Seq. I.D. # 112) H- His Ser Lys Arg Arg Leu Ile D-Psa OH (Seq. I.D. # 113) H- His Ala Lys Arg Arg Leu Ile Dhp OH (Seq. I.D. # 114) H- His Ser Lys Arg Arg Leu Ile Dhp OH (Seq. I.D. # 115) H- His Ala Lys Arg Arg Leu Ile Pheol (Seq. I.D. # 116) H- His Ser Lys Arg Arg Leu Ile Pheol (Seq. I.D. # 117) H- Ala- Ala- Abu- Arg- Arg- Leu- Ile- pFPhe -NH2 (Seq. I.D. # 118) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- pFPhe -NH2 (Seq. I.D. # 119) H- Ala- Ala- Lys- Arg- Cit- Leu- Ile- pFPhe -NH2 (Seq. I.D. # 120) H- Ala- Ala- Lys- Arg- Arg- Leu- Ala- pFPhe -NH2 (Seq. I.D. # 121) H- Ala- Ala- Abu- Arg- Ser- Leu- Ile- pFPhe -NH2 (Seq. I.D. # 122) H- Ala- Ala- Lys- Gln- Arg- Leu- Ile- pFPhe -NH2 (Seq. I.D. # 123) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- pFPhe -NH2 (Seq. I.D. # 124) H- Gly- Ala- Lys- Arg- Arg- Leu- Ile- pFPhe -NH2 (Seq. I.D. # 125) H- Ala- Ala- Lys- hArg- Arg- Leu- Ile- pFPhe -NH2 (Seq. I.D. # 126) H- Ala- Ala- Lys- Ser- Arg- Leu- Ile- pFPhe -NH2 (Seq. I.D. # 127) H- Ala- Ala- Lys- Hse- Arg- Leu- Ile- pFPhe -NH2 (Seq. I.D. # 128) H- Ala- Ala- Lys- Arg- Lys- Leu- Ile- pFPhe -NH2 (Seq. I.D. # 129) H- Ala- Ala- Lys- Arg- Orn- Leu- Ile- pFPhe -NH2 (Seq. I.D. # 130) H- Ala- Ala- Lys- Arg- Gln- Leu- Ile- pFPhe -NH2 (Seq. I.D. # 131) H- Ala- Ala- Lys- Arg- Hse- Leu- Ile- pFPhe -NH2 (Seq. I.D. # 132) H- Ala- Ala- Lys- Arg- Thr- Leu- Ile- pFPhe -NH2 (Seq. I.D. # 133) H- Ala- Ala- Lys- Arg- Nva- Leu- Ile- pFPhe -NH2 (Seq. I.D. # 134) H- Ala- Ala- Lys- Arg- Arg- Phg- Ile- pFPhe -NH2 (Seq. I.D. # 135) H- Ala- Ala- Lys- Arg- Arg- Met- Ile- pFPhe -NH2 (Seq. I.D. # 136) H- Ala- Ala- Lys- Arg- Arg- Ala- Ile- pFPhe -NH2 (Seq. I.D. # 137) H- Ala- Ala- Lys- Arg- Arg- Hof- Ile- pFPhe -NH2 (Seq. I.D. # 138) H- Ala- Ala- Lys- Arg- Arg- hLeu- Ile- pFPhe -NH2 (Seq. I.D. # 139) H- Ala- Ala- Lys- Arg- Arg- alle- Ile- pFPhe -NH2 (Seq. I.D. # 140) H- Ala- Ala- Lys- Arg- Arg- Leu- Gly- pFPhe -NH2 (Seq. I.D. # 141) H- Ala- Ala- Lys- Arg- Arg- Leu- βAla pFPhe -NH2 (Seq. I.D. # 142) H- Ala- Ala- Lys- Arg- Arg- Leu- Phg- pFPhe -NH2 (Seq. I.D. # 143) H- Ala- Ala- Lys- Arg- Arg- Leu- Aib- pFPhe -NH2 (Seq. I.D. # 144) H- Ala- Ala- Lys- Arg- Arg- Leu- Sar- pFPhe -NH2 (Seq. I.D. # 145) H- Ala- Ala- Lys- Arg- Arg- Leu- Pro- pFPhe -NH2 (Seq. I.D. # 146) H- Ala- Ala- Lys- Arg- Arg- Leu- Bug- pFPhe -NH2 (Seq. I.D. # 147) H- Ala- Ala- Lys- Arg- Arg- Leu- Ser- pFPhe -NH2 (Seq. I.D. # 148) H- Ala- Ala- Lys- Arg- Arg- Leu- Asp- pFPhe -NH2 (Seq. I.D. # 149) H- Ala- Ala- Lys- Arg- Arg- Leu- Asn- pFPhe -NH2 (Seq. I.D. # 150) H- Ala- Ala- Lys- Arg- Arg- Leu- pFPhe- Phe -NH2 (Seq. I.D. # 151) H- Ala- Ala- Lys- Arg- Arg- Leu- diClPhe Phe -NH2 (Seq. I.D. # 152) H- Ala- Ala- Lys- Arg- Arg- Leu- pClPhe- Phe -NH2 (Seq. I.D. # 153) H- Ala- Ala- Lys- Arg- Arg- Leu- mClPhe Phe -NH2 (Seq. I.D. # 154) H- Ala- Ala- Lys- Arg- Arg- Leu- oClPhe- Phe -NH2 (Seq. I.D. # 155) H- Ala- Ala- Lys- Arg- Arg- Leu- pIPhe- Phe -NH2 (Seq. I.D. # 156) H- Ala- Ala- Lys- Arg- Arg- Leu- TyrMe- Phe -NH2 (Seq. I.D. # 157) H- Ala- Ala- Lys- Arg- Arg- Leu- Thi- Phe -NH2 (Seq. I.D. # 158) H- Ala- Ala- Lys- Arg- Arg- Leu- Pya- Phe -NH2 (Seq. I.D. # 159) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- diClPhe -NH2 (Seq. I.D. # 160) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- pClPhe -NH2 (Seq. I.D. # 161) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- mClPhe -NH2 (Seq. I.D. # 162) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- oClPhe -NH2 (Seq. I.D. # 163) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- Phg -NH2 (Seq. I.D. # 164) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- TyrMe -NH2 (Seq. I.D. # 165) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- Thi -NH2 (Seq. I.D. # 166) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- Pya -NH2 (Seq. I.D. # 167) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- Inc -NH2 (Seq. I.D. # 168)

and the cyclic peptides: 5,8-cyclo-[H-His-Ala-Lys-Arg-Lys-Leu-Phe-Gly] (Seq. I.D. # 169) 5,8-cyclo-[H-His-Ala-Lys-Arg-Orn-Leu-Phe-Gly] (Seq. I.D. # 170)


26. A peptide of the formula III or IV; H′X₂K′R₁R₂L′X₅F (formula III) (SEQ ID No. ______) or H′X₂K′R₁R₂L′FX₅  (formula IV) (SEQ ID No. ______) or a variant thereof, wherein H′ is nothing, His, D-His, Ala, Thi, Hse, Phe, or Dab; X₂ is Ala, Ser, Abu, Val; K′ is Lys, Arg, or Abu; R₁ is Arg, Lys, or Gln; and R₂ is Arg, forms a cyclic peptide with the C-terminal residue, Ser, or Cit; L′ is Leu or Ile; X₅ is Ile, Leu, Gly, or Ala; and F′ is Phe, para-fluoroPhe, meta-fluoroPhe, L-Psa, 2-Nap,Dhp, or D-Psa.
 27. A peptide according to claim 26, wherein X₂ is alanine.
 28. A peptide according to claim 26, wherein X₅ isoleucine.
 29. A peptide according to claim 26 of the formula IV H′X₂K′R₁R₂L° F′X₅  (SEQ ID No. ______).
 30. The peptide of claim 26, wherein the peptide is in a cyclic form by virtue of a linkage between the C-terminal residue and the residue 3 upstream to it.
 31. A peptide according to claim 30, wherein X₂ is Ala and X₅ is Ile.
 32. A peptide according to claim 26, wherein F′ is para-fluoro-Phe and H′ is Ala or nothing.
 33. The peptide of claim 26, wherein K′ is Abu; R₁ is Gln; R₂ is Cit or Ser; and X₅ is Ala.
 34. A peptide according to claim 26 selected from the group consisting of: H- his- Ala- Lys- Arg- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # ?) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- Phe- -NH2 (Seq. I.D. # 172) H- Ala- Lys- Arg- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 173) H- Thi- Ala- Lys- Arg- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 174) H- Hse- Ala- Lys- Arg- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 175) H- Phe- Ala- Lys- Arg- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 176) H- Dab- Ala- Lys- Arg- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 177) H- His- Abu- Lys- Arg- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 178) H- His- Val- Lys- Arg- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 179) H- His- Ala- Arg- Arg- Arg- Leu- Ile- Phe -NH2 (Seq. I.D. # 180) H- His- Ala- Lys- Arg- Arg- Ile- Ile- Phe -NH2 (Seq. I.D. # 181) H- His- Ala- Lys- Arg- Arg- Leu- Leu- Phe -NH2 (Seq. I.D. # 182) H- His- Ala- Lys- Arg- Arg- Leu- Ile- pFPhe -NH2 (Seq. I.D. # ?) H- His- Ala- Lys- Arg- Arg- Leu- Ile- 2Nap -NH2 (Seq. I.D. # 103) H- His Ala Lys Arg Arg Leu Ile D-Psa OH (Seq. I.D. # 116) H- His Ser Lys Arg Arg Leu Ile Dhp OH (Seq. I.D. # 117) H- Ala- Ala- Abu- Arg- Arg- Leu- Ile- pFPhe -NH2 (Seq. I.D. # 118) H- Ala- Ala- Lys- Arg- Arg- Leu- Ile- pFPhe -NH2 (Seq. I.D. # 119) H- Ala- Ala- Lys- Arg- Cit- Leu- Ile- pFPhe -NH2 (Seq. I.D. # 120) H- Ala- Ala- Lys- Arg- Arg- Leu- Ala- pFPhe -NH2 (Seq. I.D. # 121) H- Ala- Ala- Abu- Arg- Ser- Leu- Ile- pFPhe -NH2 (Seq. I.D. # 122) H- Ala- Ala- Lys- Gln- Arg- Leu- Ile- pFPhe -NH2 (Seq. I.D. # 123) H- Ala- Lys- Arg- Arg- Leu- Ile- pFPhe -NH2 (Seq. I.D. # 183)


35. An assay for identifying candidate substances capable of binding to a cyclin associated with a G1 control CDK enzyme and/or inhibition of said enzyme, comprising; (a) bringing into contact i) a p21 derived peptide as defined in claim 1, ii) said cyclin or portion thereof or cyclin groove, iii) said CDK or portion thereof and iv) said candidate substance, under conditions wherein, in the absence of the candidate substance being an inhibitor of the cyclin/CDK interaction, the p21 derived peptide would bind to said cyclin or portion thereof or cyclin groove, and (b) monitoring any change in the expected binding of the p21 derived peptide and the cyclin or portion thereof or cyclin groove.
 36. An assay for the identification of compounds that interact with a cyclin or a cyclin when complexed with the physiologically relevant CDK, comprising; (a) incubating a candidate compound and peptide of formula I; X₁X₂X₃RX₄LX₅F  (formula II) (SEQ ID No. 2) wherein X₁, X₃, X₄ and X₅ may be any amino acid and X₂ is serine or alanine; and variants thereof or a peptide of the formula III or IV: H′X′₂K′R₁R₂L′X′₅F′ (formula III) (SEQ ID No. ______) or H′X′₂K′R₁R₂L° F′X′₅ (formula IV) (SEQ ID No. ______) or a variant thereof, wherein H′ is His, nothing, D-His, Ala, Thi, Hse, Phe, or Dab; X′₂ is Ala, Ser, Abu, Val; K′ is Lys, Arg, or Abu; R₁ is Arg, Lys, or Gln; and R₂ is Arg, forms a cyclic peptide with the C-terminal residue, Ser, or Cit; L′ is Leu or Ile; X′₅ is Ile, Leu, Gly, or Ala; F′ is Phe, para-fluoroPhe, meta-fluoroPhe, L-Psa, 2-Nap,Dhp, or D-Psa. and a cyclin or cyclin/CDK complex; (b) detecting binding of either the candidate compound or the peptide of formula II or III with cyclin.
 37. An assay for candidate compounds that interact with a cyclin by virtue of forming associations with at least two of the amino acids corresponding to the cyclin A amino acids L253, I206 and R211.
 38. An assay according to claim 37, wherein the candidate compound additionally forms associations with at least one of the amino acids corresponding to the cyclin A amino acids E223, E224, D284, D283, L253, I206 and R211.
 39. An assay according to claim 37, wherein the candidate additionally forms associations with at least one of the amino acids corresponding to the cyclin A amino acids W217, V219, V221, S408, E411, Y225, I213, L214, G257, R250, Q254, T207 and L214.
 40. An assay according to claim 37, wherein the candidate compound additionally forms associations with at least one of the amino acids corresponding to the cyclin A amino acids G222, Y225, 1281, E223, E220, V279, A212, V215, L218, Q406, S408, M210, L253, L218, 1239, V256 and M200.
 41. An assay according to claim 35, wherein the cyclin is selected from cyclin A, cyclin E or cyclin D.
 42. An assay according to claim 41 wherein the cyclin is cyclin A.
 43. An assay according to claim 35, comprising use of a three dimensional model of a cyclin and a candidate compound.
 44. An assay according to claim 35, wherein at least one of the assay components is bound to a solid phase.
 45. An assay according to claim 44, wherein the p21 derived peptide is labeled such as to emit a signal when bound to said cyclin.
 46. An assay according to claim 44, wherein the cyclin is labeled such as to emit a signal when bound to the p21 derived peptide.
 47. An assay according to claim 45, wherein one of the assay components is labeled with a fluorescence emitter and the signal is detected using fluorescence polarization techniques.
 48. A method of using a cyclin in a drug screening assay comprising: (a) selecting a candidate compound by performing rational drug design with a three-dimensional model of said cyclin, wherein said selecting is performed in conjunction with computer modeling; (b) contacting the candidate compound with the cyclin; and (c) detecting the binding affinity of the candidate compound for the cyclin groove; wherein a potential drug is selected on the basis of its having a greater affinity for the cyclin groove than that of a peptide of formula II; X₁X₂X₃RX₄LX₅F  (formula II) (SEQ ID No. 2) wherein X₁, X₃, X₄ and X₅ may be any amino acid and X₂ is serine or alanine; and variants thereof or a peptide of formula III or IV: H′X′₂K′R₁R₂L′X′₅F′ (formula III) (SEQ ID No. ______) or H′X′₂K′R₁R₂L° F′X′₅ (formula IV) (SEQ ID No. ______) or a variant thereof, wherein H′ is His, nothing, D-His, Ala, Thi, Hse, Phe, or Dab; X′₂ is Ala, Ser, Abu, Val; K′ is Lys, Arg, or Abu; R₁ is Arg, Lys, or Gln; and R₂ is Arg, forms a cyclic peptide with the C-terminal residue, Ser, or Cit; L′ is Leu or Ile; X′₅ is Ile, Leu, Gly, or Ala; F′ is Phe, para-fluoroPhe, meta-fluoroPhe, L-Psa, 2-Nap,Dhp, or D-Psa.
 49. A method of using a cyclin in a drug screening assay comprising: (a) selecting a candidate compound by performing rational drug design with a three-dimensional model of said cyclin, wherein said selecting is performed in conjunction with computer modeling; (b) contacting the candidate compound with the cyclin; and (c) detecting whether said the candidate compound forms associations with at least the amino acids corresponding to the cyclin A amino acids L253, 1206 and R211.
 50. A method according to claim 49, further comprising detection of whether the candidate compound additionally forms associations with at least one of the amino acids corresponding to the cyclin A amino acids E223, E224, D284, D283, L253, I206 and R211.
 51. A method according to claim 50, further comprising detection of whether the candidate compound additionally forms associations with at least one of the amino acids corresponding to the cyclin A amino acids W217, V219, V221, S408, E411, Y225, I213, L214, G257, R250, Q254, T207 and L214.
 52. A method according to claim 50, further comprising detection of whether the candidate compound additionally forms associations with at least one of the amino acids corresponding to the cyclin A amino acids G222, Y225, I281, E223, E220, V279, A212, V215, L218, Q406, S408, M210, L253, L218, I239, V256 and M200.
 53. An assay for identifying candidate substances capable of inhibiting CDK in a cell, comprising; (a) contacting a cell comprising a cyclin or portion thereof or cyclin groove, and a CDK or portion thereof, with a candidate substance under conditions where, in the absence of the candidate substance, the cyclin or portion thereof or cyclin groove and CDK or portion thereof would interact, and (b) monitoring any change in the activity of the CDK or portion thereof, wherein inhibition of CDK activity is indicated by one or more of: G0 and/o G1/S cell cycle arrest; cell cycle-related apoptosis; suppression of E2F transcription factor activity; hypophosphorylation of cellular pRb; and in vitro anti-proliferative effects.
 54. Use of a peptide defined in claim 1 in the preparation of a medicament for use in (a) inhibition of CDK2 or (b) in the treatment of proliferative disorders such as cancers and leukaemias where inhibition of CDK2 would be beneficial. 